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1.  Profiling of the Tox21 Chemical Collection for Mitochondrial Function to Identify Compounds that Acutely Decrease Mitochondrial Membrane Potential 
Background: Mitochondrial dysfunction has been implicated in the pathogenesis of a variety of disorders including cancer, diabetes, and neurodegenerative and cardiovascular diseases. Understanding whether different environmental chemicals and druglike molecules impact mitochondrial function represents an initial step in predicting exposure-related toxicity and defining a possible role for such compounds in the onset of various diseases.
Objectives: We sought to identify individual chemicals and general structural features associated with changes in mitochondrial membrane potential (MMP).
Methods: We used a multiplexed [two end points in one screen; MMP and adenosine triphosphate (ATP) content] quantitative high throughput screening (qHTS) approach combined with informatics tools to screen the Tox21 library of 10,000 compounds (~ 8,300 unique chemicals) at 15 concentrations each in triplicate to identify chemicals and structural features that are associated with changes in MMP in HepG2 cells.
Results: Approximately 11% of the compounds (913 unique compounds) decreased MMP after 1 hr of treatment without affecting cell viability (ATP content). In addition, 309 compounds decreased MMP over a concentration range that also produced measurable cytotoxicity [half maximal inhibitory concentration (IC50) in MMP assay/IC50 in viability assay ≤ 3; p < 0.05]. More than 11% of the structural clusters that constitute the Tox21 library (76 of 651 clusters) were significantly enriched for compounds that decreased the MMP.
Conclusions: Our multiplexed qHTS approach allowed us to generate a robust and reliable data set to evaluate the ability of thousands of drugs and environmental compounds to decrease MMP. The use of structure-based clustering analysis allowed us to identify molecular features that are likely responsible for the observed activity.
Citation: Attene-Ramos MS, Huang R, Michael S, Witt KL, Richard A, Tice RR, Simeonov A, Austin CP, Xia M. 2015. Profiling of the Tox21 chemical collection for mitochondrial function to identify compounds that acutely decrease mitochondrial membrane potential. Environ Health Perspect 123:49–56; http://dx.doi.org/10.1289/ehp.1408642
doi:10.1289/ehp.1408642
PMCID: PMC4286281  PMID: 25302578
2.  Mitochondria, Energetics, Epigenetics, and Cellular Responses to Stress 
Environmental Health Perspectives  2014;122(12):1271-1278.
Background: Cells respond to environmental stressors through several key pathways, including response to reactive oxygen species (ROS), nutrient and ATP sensing, DNA damage response (DDR), and epigenetic alterations. Mitochondria play a central role in these pathways not only through energetics and ATP production but also through metabolites generated in the tricarboxylic acid cycle, as well as mitochondria–nuclear signaling related to mitochondria morphology, biogenesis, fission/fusion, mitophagy, apoptosis, and epigenetic regulation.
Objectives: We investigated the concept of bidirectional interactions between mitochondria and cellular pathways in response to environmental stress with a focus on epigenetic regulation, and we examined DNA repair and DDR pathways as examples of biological processes that respond to exogenous insults through changes in homeostasis and altered mitochondrial function.
Methods: The National Institute of Environmental Health Sciences sponsored the Workshop on Mitochondria, Energetics, Epigenetics, Environment, and DNA Damage Response on 25–26 March 2013. Here, we summarize key points and ideas emerging from this meeting.
Discussion: A more comprehensive understanding of signaling mechanisms (cross-talk) between the mitochondria and nucleus is central to elucidating the integration of mitochondrial functions with other cellular response pathways in modulating the effects of environmental agents. Recent studies have highlighted the importance of mitochondrial functions in epigenetic regulation and DDR with environmental stress. Development and application of novel technologies, enhanced experimental models, and a systems-type research approach will help to discern how environmentally induced mitochondrial dysfunction affects key mechanistic pathways.
Conclusions: Understanding mitochondria–cell signaling will provide insight into individual responses to environmental hazards, improving prediction of hazard and susceptibility to environmental stressors.
Citation: Shaughnessy DT, McAllister K, Worth L, Haugen AC, Meyer JN, Domann FE, Van Houten B, Mostoslavsky R, Bultman SJ, Baccarelli AA, Begley TJ, Sobol RW, Hirschey MD, Ideker T, Santos JH, Copeland WC, Tice RR, Balshaw DM, Tyson FL. 2014. Mitochondria, energetics, epigenetics, and cellular responses to stress. Environ Health Perspect 122:1271–1278; http://dx.doi.org/10.1289/ehp.1408418
doi:10.1289/ehp.1408418
PMCID: PMC4256704  PMID: 25127496
3.  A systematic study of mitochondrial toxicity of environmental chemicals using quantitative high throughput screening 
Chemical research in toxicology  2013;26(9):1323-1332.
A goal of the Tox21 program is to transit toxicity testing from traditional in vivo models to in vitro assays that assess how chemicals affect cellular responses and toxicity pathways. A critical contribution of the NIH Chemical Genomics center (NCGC) to the Tox21 program is the implementation of a quantitative high throughput screening (qHTS) approach, using cell- and biochemical-based assays to generate toxicological profiles for thousands of environmental compounds. Here, we evaluated the effect of chemical compounds on mitochondrial membrane potential in HepG2 cells by screening a library of 1,408 compounds provided by the National Toxicology Program (NTP) in a qHTS platform. Compounds were screened over 14 concentrations, and results showed that 91 and 88 compounds disrupted mitochondrial membrane potential after treatment for one or five h, respectively. Seventy-six compounds active at both time points were clustered by structural similarity, producing 11 clusters and 23 singletons. Thirty-eight compounds covering most of the active chemical space were more extensively evaluated. Thirty-six of the 38 compounds were confirmed to disrupt mitochondrial membrane potential using a fluorescence plate reader and 35 were confirmed using a high content imaging approach. Among the 38 compounds, 4 and 6 induced LDH release, a measure of cytotoxicity, at 1 or 5 h, respectively. Compounds were further assessed for mechanism of action (MOA) by measuring changes in oxygen consumption rate, which enabled identification of 20 compounds as uncouplers. This comprehensive approach allows for evaluation of thousands of environmental chemicals for mitochondrial toxicity and identification of possible MOAs.
doi:10.1021/tx4001754
PMCID: PMC4154066  PMID: 23895456
mitochondrial membrane potential assay; mitochondrial toxicity; NTP 1408 compound library; oxygen consumption rate; qHTS; Tox21 collaboration
4.  The Tox21 robotic platform for assessment of environmental chemicals - from vision to reality 
Drug discovery today  2013;18(0):716-723.
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.
doi:10.1016/j.drudis.2013.05.015
PMCID: PMC3771082  PMID: 23732176
10K compound library; in vitro assays; quantitative high-throughput screening; robotic platform; Tox21 collaboration; toxicity testing
5.  Profiling of the Tox21 10K compound library for agonists and antagonists of the estrogen receptor alpha signaling pathway 
Scientific Reports  2014;4:5664.
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.
doi:10.1038/srep05664
PMCID: PMC4092345  PMID: 25012808
6.  Bisphenol A affects androgen receptor function via multiple mechanisms 
Chemico-biological interactions  2013;203(3):556-564.
Bisphenol A (BPA), is a well-known endocrine disruptor compound (EDC) that affects the normal development and function of the female and male reproductive system, however the mechanisms of action remain unclear. To investigate the molecular mechanisms of how BPA may affect ten different nuclear receptors, stable cell lines containing individual nuclear receptor ligand binding domain (LBD)-linked to the β-Gal reporter were examined by a quantitative high throughput screening (qHTS) format in the Tox21 Screening Program of the NIH. The results showed that two receptors, estrogen receptor alpha (ERα) and androgen receptor (AR), are affected by BPA in opposite direction. To confirm the observed effects of BPA on ERα and AR, we performed transient transfection experiments with full-length receptors and their corresponding response elements linked to luciferase reporters. We also included in this study two BPA analogs, bisphenol AF (BPAF) and bisphenol S (BPS). As seen in African green monkey kidney CV1 cells, the present study confirmed that BPA and BPAF act as ERα agonists (half maximal effective concentration EC50 of 10-100 nM) and as AR antagonists (half maximal inhibitory concentration IC50 of 1-2 μM). Both BPA and BPAF antagonized AR function via competitive inhibition of the action of synthetic androgen R1881. BPS with lower estrogenic activity (EC50 of 2.2 μM), did not compete with R1881 for AR binding, when tested at 30 μM. Finally, the effects of BPA were also evaluated in a nuclear translocation assays using EGPF-tagged receptors. Similar to 17β-estradiol (E2) which was used as control, BPA was able to enhance ERα nuclear foci formation but at a 100-fold higher concentration. Although BPA was able to bind AR, the nuclear translocation was reduced. Furthermore, BPA was unable to induce functional foci in the nuclei and is consistent with the transient transfection study that BPA is unable to activate AR.
doi:10.1016/j.cbi.2013.03.013
PMCID: PMC3722857  PMID: 23562765
Bisphenol A and related compounds; androgen receptor; qHTS; transfection; imaging analysis
7.  Perspectives on Validation of High-Throughput Assays Supporting 21st Century Toxicity Testing1 
ALTEX  2013;30(1):51-56.
Summary
In vitro, high-throughput screening (HTS) assays are seeing increasing use in toxicity testing. HTS assays can simultaneously test many chemicals, but have seen limited use in the regulatory arena, in part because of the need to undergo rigorous, time-consuming formal validation. Here we discuss streamlining the validation process, specifically for prioritization applications in which HTS assays are used to identify a high-concern subset of a collection of chemicals. The high-concern chemicals could then be tested sooner rather than later in standard guideline bioassays. The streamlined validation process would continue to ensure the reliability and relevance of assays for this application. We discuss the following practical guidelines: (1) follow current validation practice to the extent possible and practical; (2) make increased use of reference compounds to better demonstrate assay reliability and relevance; (3) deemphasize the need for cross-laboratory testing, and; (4) implement a web-based, transparent and expedited peer review process.
PMCID: PMC3934015  PMID: 23338806
Validation; in vitro; high-throughput screening
8.  Prediction of Cytochrome P450 Profiles of Environmental Chemicals with QSAR Models Built from Drug-like Molecules 
Molecular informatics  2012;31(11-12):783-792.
The human cytochrome P450 (CYP) enzyme family is involved in the biotransformation of many xenobiotics. As part of the U.S. Tox21 Phase I effort, we profiled the CYP activity of approximately three thousand compounds, primarily those of environmental concern, against human CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 isoforms in a quantitative high throughput screening (qHTS) format. In order to evaluate the extent to which computational models built from a drug-like library screened in these five CYP assays under the same conditions can accurately predict the outcome of an environmental compound library, five support vector machines (SVM) models built from over 17,000 drug-like compounds were challenged to predict the CYP activities of the Tox21 compound collection. Although a large fraction of the test compounds fall outside of the applicability domain (AD) of the models, as measured by k-nearest neighbor (k-NN) similarities, the predictions were largely accurate for CYP1A2, CYP2C9, and CYP3A4 ioszymes with area under the receiver operator characteristic curves (AUC-ROC) ranging between 0.82 and 0.84. The lower predictive power of the CYP2C19 model (AUC-ROC = 0.76) is caused by experimental errors and that of the CYP2D6 model (AUC-ROC = 0.76) can be rescued by rebalancing the training data. Our results demonstrate that decomposing molecules into atom types enhanced the coverage of the AD and that computational models built from drug-like molecules can be used to predict the ability of non-drug like compounds to interact with these CYPs.
doi:10.1002/minf.201200065
PMCID: PMC3583379  PMID: 23459712
Human CYPs; QSAR models; Predictive Capacity; SVM; Predictive Toxicology
9.  Improving the Human Hazard Characterization of Chemicals: A Tox21 Update 
Environmental Health Perspectives  2013;121(7):756-765.
Background: In 2008, the National Institute of Environmental Health Sciences/National Toxicology Program, the U.S. Environmental Protection Agency’s National Center for Computational Toxicology, and the National Human Genome Research Institute/National Institutes of Health Chemical Genomics Center entered into an agreement on “high throughput screening, toxicity pathway profiling, and biological interpretation of findings.” In 2010, the U.S. Food and Drug Administration (FDA) joined the collaboration, known informally as Tox21.
Objectives: The Tox21 partners agreed to develop a vision and devise an implementation strategy to shift the assessment of chemical hazards away from traditional experimental animal toxicology studies to one based on target-specific, mechanism-based, biological observations largely obtained using in vitro assays.
Discussion: Here we outline the efforts of the Tox21 partners up to the time the FDA joined the collaboration, describe the approaches taken to develop the science and technologies that are currently being used, assess the current status, and identify problems that could impede further progress as well as suggest approaches to address those problems.
Conclusion: Tox21 faces some very difficult issues. However, we are making progress in integrating data from diverse technologies and end points into what is effectively a systems-biology approach to toxicology. This can be accomplished only when comprehensive knowledge is obtained with broad coverage of chemical and biological/toxicological space. The efforts thus far reflect the initial stage of an exceedingly complicated program, one that will likely take decades to fully achieve its goals. However, even at this stage, the information obtained has attracted the attention of the international scientific community, and we believe these efforts foretell the future of toxicology.
doi:10.1289/ehp.1205784
PMCID: PMC3701992  PMID: 23603828
chemical hazard characterization; computational biology; high throughput testing; in vitro models; systems biology; Tox21
10.  RNA-Seq Profiling Reveals Novel Hepatic Gene Expression Pattern in Aflatoxin B1 Treated Rats 
PLoS ONE  2013;8(4):e61768.
Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1), a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG) than microarray analysis, including low copy and novel transcripts related to AFB1’s carcinogenic activity compared to feed controls (CTRL). Paired-end reads were mapped to the rat genome (Rn4) with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005) compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT’s) on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c. We find the rat transcriptome contains many previously unidentified, AFB1-responsive exons and transcripts supporting RNA-Seq’s capabilities to provide new insights into AFB1-mediated gene expression leading to hepatocellular carcinoma.
doi:10.1371/journal.pone.0061768
PMCID: PMC3632591  PMID: 23630614
11.  5-hmC in the brain is abundant in synaptic genes and shows differences at the exon-intron boundary 
Nature structural & molecular biology  2012;19(10):1037-1043.
5-hydroxymethylcytosine (5-hmC), a derivative of 5-methylcytosine (5-mC), is abundant in the brain for unknown reasons. Our goal was to characterize the genomic distribution of 5-hmC and 5-mC in human and mouse tissues. We assayed 5-hmC using glucosylation coupled with restriction enzyme digestion, and interrogation on microarrays. We detected 5-hmC enrichment in genes with synapse-related functions in both human and mouse brain. We also identified substantial tissue-specific differential distributions of these DNA modifications at the exon-intron boundary, in both human and mouse. This boundary change was mainly due to 5-hmC in the brain, but due to 5-mC in non-neural contexts. This pattern was replicated in multiple independent datasets and with single molecule sequencing. Moreover, in human frontal cortex, constitutive exons contained higher levels of 5-hmC, relative to alternatively-spliced exons. Our study suggests a novel role for 5-hmC in RNA splicing and synaptic function in the brain.
doi:10.1038/nsmb.2372
PMCID: PMC3465469  PMID: 22961382
12.  Quantitative High-Throughput Screening for Chemical Toxicity in a Population-Based In Vitro Model 
Toxicological Sciences  2012;126(2):578-588.
A shift in toxicity testing from in vivo to in vitro may efficiently prioritize compounds, reveal new mechanisms, and enable predictive modeling. Quantitative high-throughput screening (qHTS) is a major source of data for computational toxicology, and our goal in this study was to aid in the development of predictive in vitro models of chemical-induced toxicity, anchored on interindividual genetic variability. Eighty-one human lymphoblast cell lines from 27 Centre d’Etude du Polymorphisme Humain trios were exposed to 240 chemical substances (12 concentrations, 0.26nM–46.0μM) and evaluated for cytotoxicity and apoptosis. qHTS screening in the genetically defined population produced robust and reproducible results, which allowed for cross-compound, cross-assay, and cross-individual comparisons. Some compounds were cytotoxic to all cell types at similar concentrations, whereas others exhibited interindividual differences in cytotoxicity. Specifically, the qHTS in a population-based human in vitro model system has several unique aspects that are of utility for toxicity testing, chemical prioritization, and high-throughput risk assessment. First, standardized and high-quality concentration-response profiling, with reproducibility confirmed by comparison with previous experiments, enables prioritization of chemicals for variability in interindividual range in cytotoxicity. Second, genome-wide association analysis of cytotoxicity phenotypes allows exploration of the potential genetic determinants of interindividual variability in toxicity. Furthermore, highly significant associations identified through the analysis of population-level correlations between basal gene expression variability and chemical-induced toxicity suggest plausible mode of action hypotheses for follow-up analyses. We conclude that as the improved resolution of genetic profiling can now be matched with high-quality in vitro screening data, the evaluation of the toxicity pathways and the effects of genetic diversity are now feasible through the use of human lymphoblast cell lines.
doi:10.1093/toxsci/kfs023
PMCID: PMC3307611  PMID: 22268004
chemical cytotoxicity; apoptosis; HapMap; lymphoblasts; qHTS
13.  Endocrine-Disrupting Chemicals (EDCs): In Vitro Mechanism of Estrogenic Activation and Differential Effects on ER Target Genes 
Environmental Health Perspectives  2013;121(4):459-466.
Background: Endocrine-disrupting chemicals (EDCs) influence the activity of estrogen receptors (ERs) and alter the function of the endocrine system. However, the diversity of EDC effects and mechanisms of action are poorly understood.
Objectives: We examined the agonistic activity of EDCs through ERα and ERβ. We also investigated the effects of EDCs on ER-mediated target genes.
Methods: HepG2 and HeLa cells were used to determine the agonistic activity of EDCs on ERα and ERβ via the luciferase reporter assay. Ishikawa cells stably expressing ERα were used to determine changes in endogenous ER target gene expression by EDCs.
Results: Twelve EDCs were categorized into three groups on the basis of product class and similarity of chemical structure. As shown by luciferase reporter analysis, the EDCs act as ER agonists in a cell type– and promoter-specific manner. Bisphenol A, bisphenol AF, and 2-2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (group 1) strongly activated ERα estrogen responsive element (ERE)-mediated responses. Daidzein, genistein, kaempferol, and coumestrol (group 2) activated both ERα and ERβ ERE-mediated activities. Endosulfan and kepone (group 3) weakly activated ERα. Only a few EDCs significantly activated the “tethered” mechanism via ERα or ERβ. Results of real-time polymerase chain reaction indicated that bisphenol A and bisphenol AF consistently activated endogenous ER target genes, but the activities of other EDCs on changes of ER target gene expression were compound specific.
Conclusion: Although EDCs with similar chemical structures (in the same group) tended to have comparable ERα and ERβ ERE-mediated activities, similar chemical structure did not correlate with previously reported ligand binding affinities of the EDCs. Using ERα-stable cells, we observed that EDCs differentially induced activity of endogenous ER target genes.
doi:10.1289/ehp.1205951
PMCID: PMC3620735  PMID: 23384675
E2; EDCs; ERα; ERβ; ERE; ER target genes. Environ Health Perspect 121:459–466 (2013)
14.  Dose-Response Modeling of High-Throughput Screening Data 
Journal of biomolecular screening  2009;14(10):1216-1227.
The National Toxicology Program is developing a high throughput screening (HTS) program to set testing priorities for compounds of interest, to identify mechanisms of action, and potentially to develop predictive models for human toxicity. This program will generate extensive data on the activity of large numbers of chemicals in a wide variety of biochemical-and cell-based assays. The first step in relating patterns of response among batteries of HTS assays to in vivo toxicity is to distinguish between positive and negative compounds in individual assays. Here, we report on a statistical approach developed to identify compounds positive or negative in a HTS cytotoxicity assay based on data collected from screening 1353 compounds for concentration-response effects in nine human and four rodent cell types. In this approach, we develop methods to normalize the data (removing bias due to the location of the compound on the 1536-well plates used in the assay) and to analyze for concentration-response relationships. Various statistical tests for identifying significant concentration-response relationships and for addressing reproducibility are developed and presented.
doi:10.1177/1087057109349355
PMCID: PMC3471146  PMID: 19828774
high-throughput screening; dose-response; statistical modeling; viability assay
15.  Profiling Environmental Chemicals for Activity in the Antioxidant Response Element Signaling Pathway Using a High Throughput Screening Approach 
Environmental Health Perspectives  2012;120(8):1150-1156.
Background: Oxidative stress has been implicated in the pathogenesis of a variety of diseases ranging from cancer to neurodegeneration, highlighting the need to identify chemicals that can induce this effect. The antioxidant response element (ARE) signaling pathway plays an important role in the amelioration of oxidative stress. Thus, assays that detect the up-regulation of this pathway could be useful for identifying chemicals that induce oxidative stress.
Objectives: We used cell-based reporter methods and informatics tools to efficiently screen a large collection of environmental chemicals and identify compounds that induce oxidative stress.
Methods: We utilized two cell-based ARE assay reporters, β-lactamase and luciferase, to screen a U.S. National Toxicology Program 1,408-compound library (NTP 1408, which contains 1,340 unique compounds) for their ability to induce oxidative stress in HepG2 cells using quantitative high throughput screening (qHTS).
Results: Roughly 3% (34 of 1,340) of the unique compounds demonstrated activity across both cell-based assays. Based on biological activity and structure–activity relationship profiles, we selected 50 compounds for retesting in the two ARE assays and in an additional follow-up assay that employed a mutated ARE linked to β-lactamase. Using this strategy, we identified 30 compounds that demonstrated activity in the ARE-bla and ARE-luc assays and were able to determine structural features conferring compound activity across assays.
Conclusions: Our results support the robustness of using two different cell-based approaches for identifying compounds that induce ARE signaling. Together, these methods are useful for prioritizing chemicals for further in-depth mechanism-based toxicity testing.
doi:10.1289/ehp.1104709
PMCID: PMC3440086  PMID: 22551509
ARE; Nrf2; oxidative stress; qHTS; toxicity; Tox21
16.  Testing an Aflatoxin B1 Gene Signature in Rat Archival Tissues 
Chemical Research in Toxicology  2012;25(5):1132-1144.
Archival tissues from laboratory studies represent a unique opportunity to explore the relationship between genomic changes and agent-induced disease. In this study, we evaluated the applicability of qPCR for detecting genomic changes in formalin-fixed, paraffin-embedded (FFPE) tissues by determining if a subset of 14 genes from a 90-gene signature derived from microarray data and associated with eventual tumor development could be detected in archival liver, kidney, and lung of rats exposed to aflatoxin B1 (AFB1) for 90 days in feed at 1 ppm. These tissues originated from the same rats used in the microarray study. The 14 genes evaluated were Adam8, Cdh13, Ddit4l, Mybl2, Akr7a3, Akr7a2, Fhit, Wwox, Abcb1b, Abcc3, Cxcl1, Gsta5, Grin2c and C8orf46 homolog. The qPCR FFPE liver results were compared to the original liver microarray data and to qPCR results using RNA from fresh frozen liver. Archival liver paraffin blocks yielded 30 to 50 μg of degraded RNA that ranged in size from 0.1 to 4 kB. qPCR results from FFPE and fresh frozen liver samples were positively correlated (p≤0.05) by regression analysis and showed good agreement in direction and proportion of change with microarray data for 11 of 14 genes. All 14 transcripts could be amplified from FFPE kidney RNA except the glutamate receptor gene Grin2c; however, only Abcb1b was significantly upregulated from control. Abundant constitutive transcripts, S18 and β-actin, could be amplified from lung FFPE samples, but the narrow RNA size range (25–500 bp length) prevented consistent detection of target transcripts. Overall, a discrete gene signature derived from prior transcript profiling and representing cell cycle progression, DNA damage response, and xenosensor and detoxication pathways was successfully applied to archival liver and kidney by qPCR and indicated that gene expression changes in response to subchronic AFB1 exposure occurred predominantly in liver, the primary target for AFB1-induced tumors. We conclude that an evaluation of gene signatures in archival tissues can be an important toxicological tool for evaluating critical molecular events associated with chemical exposures.
doi:10.1021/tx3000945
PMCID: PMC3358548  PMID: 22545673
17.  Identification of quaternary ammonium compounds as potent inhibitors of hERG potassium channels 
Toxicology and applied pharmacology  2011;252(3):250-258.
The human ether-a-go-go-related gene (hERG) channel, a member of a family of voltage-gated potassium (K+) channels, plays a critical role in the repolarization of the cardiac action potential. The reduction of hERG channel activity as a result of adverse drug effects or genetic mutations may cause QT interval prolongation and potentially lead to acquired long QT syndrome. Thus, screening for hERG channel activity is important in drug development. Cardiotoxicity associated with the inhibition of hERG channels by environmental chemicals is also a public health concern. To assess the inhibitory effects of environmental chemicals on hERG channel function, we screened the National Toxicology Program (NTP) collection of 1408 compounds by measuring thallium influx into cells through hERG channels. Seventeen compounds with hERG channel inhibition were identified with IC50 potencies ranging from 0.26 to 22 μM. Twelve of these compounds were confirmed as hERG channel blockers in an automated whole cell patch clamp experiment. In addition, we investigated the structure-activity relationship of seven compounds belonging to the quaternary ammonium compound (QAC) series on hERG channel inhibition. Among four active QAC compounds, tetra-n-octylammonium bromide was the most potent with an IC50 value of 260 nM in the thallium influx assay and 80 nM in the patch clamp assay. The potency of this class of hERG channel inhibitors appears to depend on the number and length of their aliphatic side-chains surrounding the charged nitrogen. Profiling environmental compound libraries for hERG channel inhibition provides information useful in prioritizing these compounds for cardiotoxicity assessment in vivo.
doi:10.1016/j.taap.2011.02.016
PMCID: PMC3079779  PMID: 21362439
cardiotoxicity; hERG; long QT syndrome; NTP 1408 library; patch clamp; qHTS; tetra-n-octylammonium bromide; thallium influx
18.  Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines 
Included among the quantitative high throughput screens (qHTS) conducted in support of the U.S. Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in 7 isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis.
doi:10.1002/em.20656
PMCID: PMC3278799  PMID: 21538559
DT40 DNA repair-deficient cell lines; quantitative high throughput screens; cytotoxicity; genotoxicity; chromosomal aberrations; γH2AX positive foci
19.  Safety Assessment of Allergic Contact Dermatitis Hazards: An Analysis Supporting Reduced Animal Use for the Murine Local Lymph Node Assay 
The original Organisation for Economic Co-operation and Development Test Guideline 429 (OECD TG 429) for the murine local lymph node assay (LLNA) required five mice/group if mice were processed individually. We used data from 83 LLNA tests (275 treated groups) to determine the impact on the LLNA outcome of reducing the group size from five to four. From DPM measurements, we formed all possible four-mice and five-mice combinations for the treated and control groups. Stimulation index (SI) values from each four-mice combination were compared with those from five-mice combinations, and agreement (both SI < 3 or both SI ≥ 3) determined. Average agreement between group sizes was 97.5% for the 275 treated groups. Compared test-by-test, 90% (75/83) of the tests had 100% agreement; agreement was 83% for the remaining eight tests. Disagreement was due primarily to variability in animal responses and closeness of the SI to three (positive response threshold) rather than to group size reduction. We conclude that using four rather than five mice per group would reduce animal use by 20% without adversely impacting LLNA performance. This analysis supported the recent update to OECD TG 429 allowing a minimum of four mice/group when each mouse is processed individually.
doi:10.1016/j.yrtph.2010.10.004
PMCID: PMC3026076  PMID: 20974208
local lymph node assay; skin sensitization; alternative test method; animal reduction; sample size; OECD Test Guideline 429
21.  Genetic Toxicology in the 21st Century: Reflections and Future Directions 
A symposium at the 40th anniversary of the Environmental Mutagen Society, held from October 24–28, 2009 in St. Louis, MO, surveyed the current status and future directions of genetic toxicology. This article summarizes the presentations and provides a perspective on the future. An abbreviated history is presented, highlighting the current standard battery of genotoxicity assays and persistent challenges. Application of computational toxicology to safety testing within a regulatory setting is discussed as a means for reducing the need for animal testing and human clinical trials, and current approaches and applications of in silico genotoxicity screening approaches across the pharmaceutical industry were surveyed and are reported here. The expanded use of toxicogenomics to illuminate mechanisms and bridge genotoxicity and carcinogenicity, and new public efforts to use high-throughput screening technologies to address lack of toxicity evaluation for the backlog of thousands of industrial chemicals in the environment are detailed. The Tox21 project involves coordinated efforts of four U.S. Government regulatory/research entities to use new and innovative assays to characterize key steps in toxicity pathways, including genotoxic and nongenotoxic mechanisms for carcinogenesis. Progress to date, highlighting preliminary test results from the National Toxicology Program is summarized. Finally, an overview is presented of ToxCast™, a related research program of the U.S. Environmental Protection Agency, using a broad array of high throughput and high content technologies for toxicity profiling of environmental chemicals, and computational toxicology modeling. Progress and challenges, including the pressing need to incorporate metabolic activation capability, are summarized.
doi:10.1002/em.20653
PMCID: PMC3160238  PMID: 21538556
genotoxicity; mutagenicity; toxicogenomics; high throughput screening
22.  Chemical Genomics Profiling of Environmental Chemical Modulation of Human Nuclear Receptors 
Environmental Health Perspectives  2011;119(8):1142-1148.
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.
doi:10.1289/ehp.1002952
PMCID: PMC3237348  PMID: 21543282
assay performance; chemical genomics; cytotoxicity; nuclear receptors; qHTS; Tox21
23.  Weighted Feature Significance: A Simple, Interpretable Model of Compound Toxicity Based on the Statistical Enrichment of Structural Features 
Toxicological Sciences  2009;112(2):385-393.
In support of the U.S. Tox21 program, we have developed a simple and chemically intuitive model we call weighted feature significance (WFS) to predict the toxicological activity of compounds, based on the statistical enrichment of structural features in toxic compounds. We trained and tested the model on the following: (1) data from quantitative high–throughput screening cytotoxicity and caspase activation assays conducted at the National Institutes of Health Chemical Genomics Center, (2) data from Salmonella typhimurium reverse mutagenicity assays conducted by the U.S. National Toxicology Program, and (3) hepatotoxicity data published in the Registry of Toxic Effects of Chemical Substances. Enrichments of structural features in toxic compounds are evaluated for their statistical significance and compiled into a simple additive model of toxicity and then used to score new compounds for potential toxicity. The predictive power of the model for cytotoxicity was validated using an independent set of compounds from the U.S. Environmental Protection Agency tested also at the National Institutes of Health Chemical Genomics Center. We compared the performance of our WFS approach with classical classification methods such as Naive Bayesian clustering and support vector machines. In most test cases, WFS showed similar or slightly better predictive power, especially in the prediction of hepatotoxic compounds, where WFS appeared to have the best performance among the three methods. The new algorithm has the important advantages of simplicity, power, interpretability, and ease of implementation.
doi:10.1093/toxsci/kfp231
PMCID: PMC2777082  PMID: 19805409
modeling; toxicity prediction; structural features; cell viability; caspase-3,7 activation; in vivo toxicity
24.  Identification of Chemical Compounds that Induce HIF-1α Activity 
Toxicological Sciences  2009;112(1):153-163.
Cellular metabolism depends on the availability of oxygen and the major regulator of oxygen homeostasis is hypoxia-inducible factor 1 (HIF-1), a highly conserved transcription factor that plays an essential role in cellular and systemic homeostatic responses to hypoxia. HIF-1 is a heterodimeric transcription factor composed of hypoxia-inducible HIF-1α and constitutively expressed HIF-1β. Under hypoxic conditions, the two subunits dimerize, allowing translocation of the HIF-1 complex to the nucleus where it binds to hypoxia-response elements (HREs) and activates expression of target genes implicated in angiogenesis, cell growth, and survival. The HIF-1 pathway is essential to normal growth and development, and is involved in the pathophysiology of cancer, inflammation, and ischemia. Thus, there is considerable interest in identifying compounds that modulate the HIF-1 signaling pathway. To assess the ability of environmental chemicals to stimulate the HIF-1 signaling pathway, we screened a National Toxicology Program collection of 1408 compounds using a cell-based β-lactamase HRE reporter gene assay in a quantitative high-throughput screening (qHTS) format. Twelve active compounds were identified. These compounds were tested in a confirmatory assay for induction of vascular endothelial growth factor, a known hypoxia target gene, and confirmed compounds were further tested for their ability to mimic the effect of a reduced-oxygen environment on hypoxia-regulated promoter activity. Based on this testing strategy, three compounds (o-phenanthroline, iodochlorohydroxyquinoline, cobalt sulfate heptahydrate) were confirmed as hypoxia mimetics, whereas two compounds (7-diethylamino-4-methylcoumarin and 7,12-dimethylbenz(a)anthracence) were found to interact with HIF-1 in a manner different from hypoxia. These results demonstrate the effectiveness of qHTS in combination with secondary assays for identification of HIF-1α inducers and for distinguishing among inducers based on their pattern of activated hypoxic target genes. Identification of environmental compounds having HIF-1α activation activity in cell-based assays may be useful for prioritizing chemicals for further testing as hypoxia-response inducers in vivo.
doi:10.1093/toxsci/kfp123
PMCID: PMC2910898  PMID: 19502547
cobalt sulfate heptahydrate; 7-diethylamino-4-methylcoumarin; 7,12-dimethylbenz(a)anthracence; HIF-1α; inducers; iodochlorohydroxyquinoline; NTP 1408 compound library; o-phenanthroline; qHTS

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