- Email: [email protected]
Elucidating environmental dimensions of neurological disorders and disease: Understanding new tools from federal chemical testing programs
Science of the Total Environment 593–594 (2017) 634–640
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Elucidating environmental dimensions of neurological disorders and disease: Understanding new tools from federal chemical testing programs Jennifer McPartland a,⁎, Heather Dantzker b, Christopher Portier a a b
Environmental Defense Fund, 1875 Connecticut Ave. NW, Ste. 600, Washington, DC 20009, USA Dantzker Consulting, LLC, 2613 N. Harrison St., Arlington, VA 22207, USA
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• ToxCast/Tox21 screening data can support hypothesis-driven research. • Federal chemical screening is currently limited for identifying neurotoxicants. • Broader scientific engagement can strengthen chemical testing programs. • Use of ToxCast and Tox21 by the broader scientific community is encouraged.
a r t i c l e
i n f o
Article history: Received 25 November 2016 Received in revised form 1 March 2017 Accepted 3 March 2017 Available online xxxx Editor: Jay Gan
a b s t r a c t Background: Federal agencies are making significant investments to advance predictive approaches to evaluate chemical hazards and risks. Environmental Defense Fund (EDF) believes that engagement with the broader scientific community is critical to building and maintaining a strong biological foundation for these approaches. Objectives: On June 18–19, 2015, EDF organized a meeting to 1) foster a conversation between federal scientists advancing predictive approaches and environmental health researchers investigating environmental exposures and neurological outcomes, and 2) explore opportunities and challenges for the use of federal chemical high-throughput in vitro screening (HTS) data in hypothesis-driven research toward, ultimately, improved data for public health decision-making. Discussion: The meeting achieved its objectives. Government scientists showcased their chemical testing programs and vision for how emerging data may be used to meet agency missions. Environmental health researchers shared their experiences using federal HTS data, offered recommendations for strengthening federal HTS platforms, and expressed great interest in continued engagement with evolving federal chemical testing initiatives. Conclusions: The meeting provided an invaluable exchange between two scientific communities with a shared interest in protecting public health from harmful environmental exposures, but who have not sufficiently engaged with each other. Discussions identified opportunities and work ahead for the use of HTS data in hypothesis-driven research. Though the meeting focused on neurological outcomes, the purpose,
⁎ Corresponding author at: 1875 Connecticut Ave. NW, Ste. 600, Washington, DC 20009, USA. E-mail addresses: [email protected] (J. McPartland), [email protected] (H. Dantzker), [email protected] (C. Portier).
http://dx.doi.org/10.1016/j.scitotenv.2017.03.024 0048-9697/© 2017 Elsevier B.V. All rights reserved.
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objectives and experience of the meeting are broadly applicable. EDF strongly encourages more discourse and collaboration between federal and non-government scientists working to understand environmental influences on health outcomes. © 2017 Elsevier B.V. All rights reserved.
1. Introduction As part of a larger portfolio of federal research efforts to advance new approaches for the evaluation of chemical toxicity and risk, vast quantities of high-throughput in vitro screening (HTS) data continue to be generated by the U.S. Environmental Protection Agency's (EPA) ToxCast™ initiative (U.S. EPA, 2017a) and the Tox21 partnership led by the National Institute of Environmental Health Sciences (NIEHS)/National Toxicology Program (NTP) (Tice et al., 2013, U.S. EPA, 2017b). Though these and related federal chemical testing programs have made significant progress since their inception, more growth opportunities remain for these programs to reach their full potential of protecting public health and the environment. Three goals must be achieved to realize the promise of predictive toxicology approaches in public health decision-making: 1) development of a scientific foundation supporting their use; 2) scientific acceptance of a new testing and analysis paradigm; and 3) health-protective policies that encourage their use (McPartland et al., 2015). Achieving these goals will require contributions from the broader scientific research community, particularly those on the forefront of understanding the etiology and pathology of environmentally-influenced disease (NRC, 2007). Environmental Defense Fund (EDF), in cooperation with the U.S. EPA and NIEHS/NTP, brought federal ToxCast™ and Tox21 scientists together with environmental health researchers for a meeting entitled, Elucidating Environmental Dimensions of Neurological Disorders and Disease: Understanding New Tools from Federal Chemical Testing Programs (UC Davis Conference Center, June 18–19, 2015). The meeting's objectives included 1) fostering a conversation between government scientists and environmental health researchers on federal predictive chemical testing approaches and 2) exploring opportunities and challenges for the use of these data in hypothesis-driven research. Progress on these objectives will strengthen links between researchers and regulatory scientists, improve the use of predictive toxicology data in decision-making, and help protect public health. Neurological outcomes affect millions of people worldwide: in the U.S. alone, approximately 60,000 people are diagnosed with Parkinson's disease each year (Parkinson's Disease Foundation, 2017); the percentage of children diagnosed with attention-deficit/hyperactivity disorder (ADHD) continues to rise from 7.8% in 2003 to 11.0% in 2011 (Visser et al., 2014); and in 2012, one in 68 children were estimated to have autism spectrum disorder (ASD), a 29% increase from 2008 (Christensen et al., 2016). Since genetic factors alone have been estimated to explain only 30 to 40% of all cases of neurodevelopmental disorders (Grandjean and Landrigan, 2006), understanding the role of the environment is critical if we are to better protect public health. Elucidating the impact of environmental exposures on neurological outcomes is particularly difficult owing to the underlying biological complexity of the nervous system. Approaches like HTS hold the potential to make progress toward identifying chemical exposures that interfere with normal brain development and function.
2016a, 2017c). The meeting then focused on how environmental health researchers are already integrating these data and tools for research on neurodevelopmental (e.g., autism) and neurodegenerative (e.g., Parkinson's) outcomes. Other presenters spoke to future possibilities for these data in basic and epidemiological research. Meeting participants were given the opportunity to engage with agency scientists on the various tools available to access and query federal HTS data. Throughout the meeting, participants exchanged ideas on key opportunities and challenges related to the use of HTS data in the field of environmental health. 2. Meeting outcomes Meeting outcomes include discussions on 1) recent experiences using ToxCast™ and Tox21 data in hypothesis-driven research investigations; 2) potential applications of HTS data in future hypothesis-driven research; and 3) current barriers to the use of federal HTS data by the broader scientific community. We then discuss measures that agencies and environmental health researchers can both take to further the conversation. 2.1. ToxCast™ and Tox21 data in hypothesis-driven research: recent investigations Compared to traditional toxicity testing approaches, HTS approaches are typically faster and provide greater insight into the mechanisms by which chemicals interfere with normal biology. These efficiencies provide researchers with tools and data to screen and identify chemical targets for additional investigation. HTS data can also be combined with other data types (e.g., ‘omics’, in silico models, whole animal laboratory studies, and epidemiological data) to enrich investigations of environmental impacts on health.
1.1. Overall meeting structure
2.1.1. Identifying novel targets for vitamin D receptor signaling Dr. Seth Kullman (Department of Biological Sciences at North Carolina State University) is using data from Tox21 to examine linkages between environmental exposures, modulation of vitamin D receptor (VDR) signaling, and later life neurodevelopmental consequences. Dr. Kullman shared that vitamin D deficiency early in development can lead to neurobehavioral outcomes through, among other proposed mechanisms, interference with dopaminergic signaling (Cui et al., 2015). Using data from an 8500-compound library screened through a Tox21 human VDR assay, Kullman's team identified multiple putative VDR agonists and antagonists. Orthogonal in vitro testing (e.g., human VDR transient transactivation assays and receptor-coregulator protein interactions assays) yielded generally concordant results. The strongest agonist and antagonist candidates were further examined for interaction with zebrafish VDR and demonstrated 100% concordance with the human VDR assays. Initial whole zebrafish studies of cadmium chloride, one of the identified VDR antagonists, revealed effects on larval locomotor activity. Dr. Kullman concluded that federal HTS data have been instrumental in identifying novel targets for his team's receptor of interest (VDR) that otherwise would have not likely been discovered.
The two-day meeting opened with presentations describing the ToxCast™ and Tox21 initiatives, including descriptions of their respective assay batteries, data, and accompanying user interfaces (U.S. EPA,
2.1.2. Using HTS data to develop prediction models of chemical toxicity Dr. Heather Patisaul (Department of Biological Sciences at North Carolina State University) investigates neuroendocrine disruption
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of brain regions and behavior, including those associated with ASD. Dr. Patisaul and colleagues set out to develop a prediction model for neurodevelopmental effects using ToxCast™ HTS data. Searching the ToxCast™ assay battery for steroid hormone and neuropeptide relevant assays, the research team identified assays for vasopressin (2 receptor binding assays) and oxytocin (1 receptor binding assay). Of the 1000 + chemicals assessed in these assays, Dr. Patisaul's team found 20 positive hits, including bisphenol-A (BPA). BPA has been shown to alter oxytocin, vasopressin, and dopaminergic neuron numbers in vivo and to decrease sociality in various animal models including male prairie voles, a species noted for its prosocial behaviors (Adewale et al., 2011; Sullivan et al., 2014; Wolstenholme et al., 2011, 2013). Dr. Patisaul and colleagues determined that too few ToxCast™ assays were available to develop a robust prediction model for neurodevelopmental toxicity and turned their efforts to the development of a prediction model for endocrine activity (Filer et al., 2014). One component of their model evaluates xenobiotic interference with monoamine signaling using 13 cell-free binding assays in ToxCast. Of the 1858 chemicals evaluated only chlorpromazine hydrochloride produced appreciable activity in this component of the model, suggesting a need for additional assays that examine chemical interactions with monoamine receptors. 2.1.3. Using HTS data for relational analysis and forecasting toxicity Dr. Scott Auerbach (Biomolecular Screening Branch, National Toxicology Program, NIEHS) uses federal HTS data to prioritize chemicals for further targeted investigation. Dr. Auerbach described a multi-step, integrated bioactivity profiling approach using publically-available data mining and visualization tools (NTP, 2017a). His approach operates on the premise that chemicals that exhibit similar biological activity across HTS assays will exhibit similar in vivo biological/toxicological properties. As an illustration, Dr. Auerbach used triethyltin, a well-documented neurotoxic compound, to identify other potential neurotoxic chemicals in the Tox21 chemical library. Zinc dimethyldithiocarbamate and zinc pyrithione were identified. Additional cursory review of the literature revealed that zinc dimethyldithiocarbamate has been shown to cause motor changes when administered to rats (Hodge et al., 1956), as well as an association with Parkinson's disease (Moretto and Colosio, 2013), and that zinc pyrithione has resulted in toxicity to the peripheral nerve tissue and skeletal muscle in lab animals (SCCS, 2014). Dr. Auerbach also illustrated a “crowd-sourcing” approach that involves using chemical-to-biological annotations of nearest neighbors as ascribed in various databases (e.g., DrugBank, Toxin Target, Leadscope, PubMed MeSH terms etc.). Using zinc pyrithione as a hypothetical chemical with undefined toxicity, applying this approach yielded several biological annotations related to developmental toxicity, a finding consistent with existing data showing effects on neuronal signaling that may be related to observed paralytic effects (SCCS, 2014). 2.1.4. Using HTS data to complement zebrafish investigations of chemical toxicity Dr. Robert Tanguay (Department of Molecular and Environmental Toxicology at Oregon State University) described his efforts to identify and evaluate chemical effects on embryonic development in zebrafish (i.e., between 6 and 120 h post fertilization). Dr. Tanguay and collaborators blindly evaluated 1060 ToxCast™ chemicals in: 1) an embryonic photomotor response assay that examines changes in a well-defined embryonic photomotor response to high-intensity light (Reif et al., 2016); 2) a larval locomotor response assay that measures changes in swimming behavior during alternating periods of dark and light; and 3) an assay for aberrations in 22 distinct morphological endpoints (e.g., notochord, yolk sac) (Truong et al., 2014). They found 376 active compounds in the embryonic photomotor response assay, many exhibiting non-monotonic dose-response curves.
Embryonic photomotor response appeared to be predictive of morphological teratogenic outcomes observed four days later. Dr. Tanguay emphasized that HT in vivo assays (e.g., zebrafish) can provide rapid data for chemical prioritization, structure-based prediction models, and phenotypic anchoring for adverse outcome pathway discovery. Dr. Francisco Quintana (Department of Neurology at Brigham and Women's Hospital and Harvard Medical School) is interested in understanding how environmental influences may contribute to inflammation-induced neurodegeneration in multiple sclerosis (MS). Dr. Quintana focuses on immune system drivers of progressive loss of neuronal myelin in both relapsing-remitting MS—driven largely by abnormal immune activity by effector (pathogenic) T cells—as well as secondary progressive MS, driven largely by astrocytes (Farez et al., 2015, Mayo et al., 2014, Rothhammer et al., 2016). Dr. Quintana and collaborators used ToxCast™ data measuring bioactivity against biological targets associated with autoimmune disease pathways (i.e., AHR, TNF, IFN, STAT3, TGFb1, IL1a, JAK2) to select compounds for screening in a zebrafish model of T-cell dependent inflammatory bowel disease (IBD) (McGovern et al., 2015). He explained that T cells that cause pathology in IBD share many features with T cells that cause pathology in MS (Marson et al., 2015). Eighty-five ToxCast™ compounds of interest were screened in the zebrafish model of colitis with 24 compounds showing significant inflammatory or anti-inflammatory effects. Four of these (2 inflammatory and 2 anti-inflammatory) have been selected for additional studies in mice. 2.2. ToxCast™ and Tox21 data in hypothesis-driven research: Potential future applications Looking forward, meeting participants explored how federal HTS data may be used in future research. 2.2.1. Using federal HTS data to inform epidemiological investigations Dr. Newschaffer (AJ Drexel Autism Institute, Drexel University) described how epidemiological approaches could employ large HTS data sets to 1) identify additional ASD exposure candidates and 2) help create candidate ASD ‘subexposomes’ from large exposomic data sets. He explained that HTS data could broaden the scope of candidate chemicals for epidemiological research and that perturbations of biological pathways identified through HTS could be interpreted as signals to be measured in human biological samples. One such opportunity described by Dr. Newschaffer is in Genome-Environment Wide Interaction Studies (GEWIS), whereby chemical HTS data could be used to identify exposures to measure and assess. As an example, he highlighted the ongoing Study to Explore Early Development (SEED) case-control investigation—currently one of the largest studies in the U.S. to identify environmental and other factors that put children at risk for ASD (Schendel et al., 2012). Dr. Tanner (Department of Neurology, University of California, San Francisco, and Parkinson's Disease Research, Education, and Clinical Center, San Francisco Veterans Affairs Health Care System) provided several examples of how HTS data could be useful for PD environmental epidemiology. The street drug, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes PD-like symptoms in people (Calne and Langston, 1983). Mining HTS data for compounds with similar mechanistic characteristics as MPTP would help identify other potential environmental contributors to PD. For compounds identified in human studies of PD (e.g., paraquat and 2,4-D), such a database could provide additional insight into their mechanisms of action considering what is known and unknown about PD etiology and pathogenesis. As a final example, Dr. Tanner highlighted that in her earlier investigations of farmworkers and PD (Tanner et al., 2011), access to HTS mechanistic data would have facilitated consideration of a broader set of compounds to investigate beyond the pesticides she studied. Because gene–environment interaction appears to be important to the etiology of PD (Goldman et al., 2012), databases integrating
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genetic information and pathways for xenobiotic metabolism would be especially helpful. 2.2.2. Research approaches using HTS data to address neurological health outcomes Meeting participants outlined several research proposals for how HTS data can be integrated into hypothesis-driven research related to neurological disease outcomes, and specifically how federal HTS assay batteries could be further augmented toward this end. One proposal was to mine federal HTS datasets to identify chemicals that may impact attention, specifically ADHD, by searching for chemicals structurally and biologically (i.e., shared bioactivity in the HTS assays) similar to polybrominated diphenyl ethers (PBDEs)—a chemical class associated with neurological effects including effects on attention (Berghuis et al., 2015, Cowell et al., 2015, Sagiv et al., 2015). Identified compounds would be assessed in zebrafish for methylomic and transcriptomic effects resulting from embryonic exposures to select an even smaller subset for testing in mammalian models of attention, and eventually epidemiological studies, assuming biomarkers for these compounds exist or could be developed. A similar approach was highlighted as potentially useful for investigations of PD. Here however, more emphasis was placed on mining the federal HTS data for chemicals with shared bioactivity profiles since canonical, structurally-similar compounds associated with PD, like paraquat and MPTP, operate through very different mechanisms. Again, promising compounds would be further examined in animal and ultimately epidemiological studies. Another proposal focused on broadening the NTP's S1500 transcriptomic project, part of the larger Tox21 initiative, that seeks to measure chemically-induced changes in gene expression across a sentinel set of 1500 environmentally-responsive genes (NTP, 2017b) to identify environmental exposures related to autism. Specifically, full transcriptomic sequencing in relevant neurons could be done and compared to extant autism gene databases such as the SFARI autism gene list (SFARI, 2017). Others suggested using federal HTS data to interpret metabolomics data from an autism case-control study on cord blood in a large, previously well-characterized cohort. A final research proposal focused on neurodegenerative disorders began with the identification of compounds in the current federal HTS libraries more likely to reach the brain based on hydrophilicity and hydrophobicity. In parallel, a set of genes relevant to neurodegenerative disease would be compiled and correlated to targets currently represented in the federal HTS assays using large, highly-curated genomic databases such as NextBio (NextBio, 2017). Data from the correlated assays would be cross-referenced against compounds identified as likely to cross the blood-brain barrier to identify a targeted set of chemicals for additional assessment. It is important to note that few of the canonical genes relevant to late onset neurodegenerative outcomes are currently represented in the federal HTS testing platforms but such assays could be developed. 2.3. Critical biological realities to consider in advancing predictive chemical testing Various, inherent biological complexities continue to challenge the development of HTS platforms for neurodevelopmental and neurodegenerative outcomes, but significant progress is being made. These include accounting for influencing risk factors such as genetic and gender susceptibilities and timing of exposure, and integrating metabolic competence into HTS assays. 2.3.1. Neuro-relevant biological targets Some assays for assessing neurological endpoints are included in the federal HTS platforms but more are needed, including: • Assays that examine pharmacological manipulation of neurotransmission through, for example, receptors for oxytocin, serotonin,
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GABA, vasopressin, glutamate, and dopamine; • inclusion of assays that interrogate pathways involved in shaping neuronal connectivity in the developing brain (e.g., dendritic morphogenesis, synapse formation, and synapse stabilization); • greater representation of ASD sentinel genes, and • greater representation of genes and pathways associated with PD including α-synuclein aggregation (e.g., PARK I, PARK IV and LRRK2), degradative pathways (e.g., proteasomal degradation and autophagy), and mitochondrial dysfunction. 2.3.2. Differential genetic susceptibility Genetic susceptibility, and other forms of individual susceptibility, are critical considerations in the evaluation of potential health effects resulting from environmental exposures. However, comprehensively accounting for these susceptibilities has proven challenging for chemical testing broadly. Dr. Pamela Lein (Department of Molecular Biosciences at the University of California, Davis) described how in vitro testing may help to obtain “first-pass” information on differential effects of chemicals on different genetic backgrounds. One approach she is developing in collaboration with Dr. Isaac Pessah (Department of Molecular Biosciences, University of California, Davis) involves evaluating in vitro chemical exposures on neurotypical neurons versus neurons expressing ASD-associated gene variants like the Fragile X premutation (FMR1 containing CGG trinucleotide repeats). The Fragile X premutation is the single most prevalent gene disorder associated with ASD (Hagerman et al., 2011; Hagerman and Hagerman, 2013; Krueger and Bear, 2011; Leehey and Hagerman, 2012). Since the FMR1 gene is X-linked, two cell subpopulations, neurotypical and FMR1 premutation, can be isolated from the same human iPSC-derived neuronal cells for in vitro testing (Liu et al., 2012). Already her collaborator, Dr. Pessah, has shown differences in calcium signaling—important in determining the extent of dendritic arborization—between these two cell sub-populations. The plan is to now screen compounds in these two neuronal subpopulations for differential effects on neuronal connectivity to better understand how specific gene-environment interactions can render some individuals more susceptible to neurodevelopmental disorders from the same environmental exposures. Dr. Valerie Hu (Department of Biochemistry and Molecular Medicine at The George Washington University) described her efforts to elucidate the sexual dimorphism of ASD, and how this has led to the development of a new HTS assay to screen for environmental contributors to ASD. Her research focuses on retinoic acid-related orphan receptor alpha (RORA). RORA is an ASD target of interest identified from large-scale microarray analyses of autism candidate genes (Nguyen et al., 2010). Dr. Hu's team has found that RORA transcriptionally regulates over 2500 potential targets, including over 400 autism risk genes (Sarachana and Hu, 2013). Moreover, previous animal studies examining RORA deficiency have revealed functional deficits and sexually dimorphic effects in line with ASD pathology and pathophysiology. Because of the hormonal sensitivity of RORA (Sarachana et al., 2011), Dr. Hu and her team are in the process of developing a high-throughput luciferase screening assay to identify endocrine disrupting chemicals that influence RORA expression. 2.3.3. Timing of exposure Timing of exposure is another critical consideration in the evaluation of potential health effects resulting from environmental exposures. However, accurately accounting for the timing of exposure can present challenges for in vitro chemical testing, particularly when evaluating complex endpoints like neurodevelopmental toxicity which involves dynamic, temporally and spatially orchestrated biological processes. Dr. Elaine Faustman (Department of Environmental and Occupational Health Sciences at the University of Washington) presented research designed to temporally anchor in vitro cellular models of development to in vivo models of development. One approach Dr. Faustman's team is pursuing uses human neuroprogenitor stem cells
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(hNPCs) to examine forebrain development, specifically formation of cortical pyramidal neurons. Dr. Faustman is using the hNPC model to identify and define transcriptomic profiles in vitro that correspond to biological activity of humans at 5 to 10 weeks of gestation. The transcriptomic profiles of hNPC cells are tracked over 21 days in vitro. Comparison of gene expression in undifferentiated hNPC (day 0) versus the fetal brain samples shows a significant degree of concordance between the in vitro and in vivo transcriptomes (Wegner et al., 2017 in press). Genes differentially expressed across time both in vitro and in vivo were enriched for gene ontology (GO) terms associated with developmental processes including brain development. Genes with differential expression uniquely in vitro were associated with GO terms related to stress signaling, and genes with differential expression uniquely in vivo were associated with GO terms related to neurotransmission. Dr. Faustman indicated that these findings were not surprising given that there is some degree of cellular stress resulting merely from growth in culture (e.g., artificial air environment) and the well-established importance of cell-cell contact occurring in vivo among specific cell populations during neurodevelopment (Wegner et al., 2017 in press). Dr. Faustman suggested that this type of exploratory, anchoring data provides critical information for interpreting HTS data in terms of understanding what aspects of brain development are being captured in a particular in vitro testing model and which are not. It may be that a chemical acts on a neurodevelopmental pathway that is active during a specific time period of brain development and if an in vitro test system does not reflect the biology occurring during that time period then the chemical effect can be missed. 2.3.4. Metabolic competence and other issues common to HTS platforms Metabolic competence (i.e., accounting for the generation of metabolites in toxicity testing), the inherent artificial conditions of in vitro testing, as well as the unique biology of neuroprogenitor cells and neurons that may be absent in the cell lines typically used in HTS platforms all represent challenges for HTS broadly. Agency scientists are at the forefront of tackling these types of challenges for the broader research community. For example, federal agencies collaborated to launch a crowdsourcing effort, “Transform Tox Testing Challenge: Innovating for Metabolism” to identify solutions to better integrate biotransformation into its HTS assays (U.S. EPA, 2016b)—an investment that benefits all those endeavoring to employ HTS in their investigations. 2.4. Opportunities for the use of HTS data by the broader scientific community 2.4.1. Access and use of HTS data and tools Genius Bar sessions at the meeting provided participants with the opportunity to explore web-based federal HTS data dashboards and related query tools (U.S. EPA, 2016a, 2017a, 2017c) alongside expert EPA and NIEHS scientists involved in their development and application. These sessions were widely regarded as one of the best features of the meeting. Participants cited the value and need for more user guidance and tutorials; additional clarity around data processing decision rules and hit calls; and other information to help researchers interpret query results and conduct further customized analyses. Communication of when updates, corrections, or additions are made to these web-based data interfaces will continue to be an important factor in increasing use of these valuable resources. 2.4.2. Opportunities for building a user community Growing an active community of federal HTS data users, which would evolve over time into a peer-support network, would benefit both research scientists as well as resource-constrained agencies. Researchers and agency scientists would mutually benefit from dedicated agency staff who could field inquiries from external users related to the emerging data, tools, and analyses. Meeting participants expressed enthusiasm for these types of activities.
Such a user community would also benefit from hands-on, interactive training opportunities. One possible approach would be to incorporate coursework on the federal HTS data and dashboards into collegiate and graduate academic programs. Training via workshops, either regional, traveling, or those in which participants would be funded to travel would also be highly valuable and desired training opportunities. 3. Discussion In the Davis meeting, EDF met its near-term objectives of 1) fostering a conversation between government scientists and environmental health researchers to strengthen emerging predictive chemical testing approaches; and 2) exploring opportunities and challenges for the use of resultant data in hypothesis-driven research. Participants examined some of the research opportunities that can be gained by introducing environmental health scientists to the ToxCast™ and Tox21 initiatives. This proved to be a win-win situation for both the researchers and the regulatory agencies. Environmental health researchers left the meeting with a much stronger understanding of federal HTS programs and how to access and utilize federal HTS data for hypothesis-driven research. Many also left with knowledge of new compounds that may be relevant to their research on neurodevelopmental and neurodegenerative disease. Over time, as these researchers gain greater understanding of disease etiology and pathogenesis, more will be known about which cellular targets are related to certain diseases, providing regulatory agencies with new knowledge that can be integrated into chemical testing platforms. While this meeting focused on neurological outcomes, it is our hope that new conversations and collaborations will emerge between agency scientists and environmental health researchers working in other disease areas. Neurodevelopment and neurodegeneration are complex, multifaceted processes that are not fully understood. The datasets being created for regulatory chemical screening have great value beyond their original purpose. As Drs. Kullman, Patisaul, Hu, and others described, federal HTS data have the potential to help basic researchers who are examining the effect of chemicals on biological processes to further illuminate how those processes relate to disease. The datasets can also provide new chemical targets for epidemiological investigations of threats to health from environmental exposures, such as described by Drs. Newschaffer and Tanner. Examining very complicated processes with multiple targets, as described by Dr. Lein, Tanguay, Auerbach and others, benefits greatly from having large, searchable chemical databases. Potential interactions of multiple chemicals altering multiple targets in a process can be hypothesized through such a database then further examined through complementary experimentation. Currently, however, ToxCast™ and Tox21 are neither comprehensive in chemical nor biological space. We encourage agency scientists to continue to be transparent about limitations in the current HTS platforms so that external scientists can assess the utility of the data and, importantly, identify opportunities to fill data gaps. A key way for investigators to help address gaps in the datasets is to learn about the screening efforts, query the HTS data to identify what is useful to them in their own hypothesis-driven research, and simultaneously, identify what may be missing in the current assay batteries. Researchers can nominate additional assays for inclusion in federal chemical screening programs using the Tox21 assay nomination process (U.S. EPA, 2017d). In parallel, we also encourage the Tox21 and ToxCast™ programs to continue to engage with basic researchers regarding the types of assays that would be most useful for identifying disease threats, as was done by the National Toxicology Program in a 2012 workshop and subsequent work on diabetes and obesity (Thayer et al., 2012, Auerbach et al., 2016). These simultaneous activities will broaden the universe of scientists collectively working to advance and accelerate federal HTS and similar efforts. The overarching take-away from the meeting is that, despite certain challenges, federal HTS data present basic researchers and
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epidemiologists with a rich opportunity for exploration, inquiry, and collaboration, which in turn presents agencies with a mechanism for advancing its chemical testing programs. By so doing, agencies can fill current gaps, resulting in better coverage of disease-relevant processes in these test systems, and, ultimately, contributing to a more robust understanding of environmental influences on disease. Competing financial interests Dr. Heather Dantzker works as a consultant to the Environmental Defense Fund. Dr. Jennifer McPartland and Dr. Christopher Portier declare they have no actual or potential competing financial interests. Acknowledgements The authors would like to thank each of the participants and presenters at EDF's June 2015 Elucidating Environmental Dimensions of Neurological Disorders and Disease: Understanding New Tools from Federal Chemical Testing Programs meeting, including the U.S. EPA and NIEHS/ NTP staff who attended and supported the meeting's Genius Bar sessions. In addition, we would like to especially thank each of the presenters who allowed us to highlight their research in this paper and provided timely review of drafts. Finally, we would like to thank Autism Speaks and the National Parkinson Foundation for providing participant travel support to the meeting and the National Toxicology Program for issuing a small number of competitive travel awards. References Adewale, H.B., Todd, K.L., Mickens, J.A., Patisaul, H.B., 2011. The impact of neonatal bisphenol-A exposure on sexually dimorphic hypothalamic nuclei in the female rat. Neurotoxicology 32:38–49. http://dx.doi.org/10.1016/j.neuro.2010.07.008. Auerbach, S., Filer, D., Reif, D., Walker, V., Holloway, A.C., Schlezinger, J., Srinivasan, S., Svoboda, D., Judson, R., Bucher, J.R., Thayer, K.A., 2016. Prioritizing environmental chemicals for obesity and diabetes outcomes research: a screening approach using ToxCast™ high-throughput data. Environ. Health Perspect. 124:1141–1154. http:// dx.doi.org/10.1289/ehp.1510456. Berghuis, S.A., Bos, A.F., Sauer, P.J., Roze, E., 2015. Developmental neurotoxicity of persistent organic pollutants: an update on childhood outcome. Arch. Toxicol. 89:687–709. http://dx.doi.org/10.1007/s00204-015-1463-3. Calne, D.B., Langston, J.W., 1983. Aetiology of Parkinson's disease. Lancet 2, 1457–1459. Christensen, D.L., Baio, J., Braun, K.V., Bilder, D., Charles, J., Constantino, J.N., et al., 2016. Prevalence and Characteristics of Autism Spectrum Disorder Among Children Aged 8 years — Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States, 2012. MMWR Surveill Summ 65:pp. 1–23. http://dx.doi.org/10.15585/ mmwr.ss6503a1 Available at: http://www.cdc.gov/mmwr/volumes/65/ss/ss6503a1. htm (Accessed 28 Feb 2017). Cowell, W.J., Lederman, S.A., Sjödin, A., Jones, R., Wang, S., Perera, F.P., et al., 2015. Prenatal exposure to polybrominated diphenyl ethers and child attention problems at 3–7 years. Neurotoxicol. Teratol. 52 (PtB):143–150. http://dx.doi.org/10.1016/j.ntt.2015. 08.009. Cui, X., Pertile, R., Liu, P., Eyles, D.W., 2015. Vitamin D regulates tyrosine hydroxylase expression: N-cadherin a possible mediator. Neuroscience 304:90–100. http://dx.doi. org/10.1016/j.neuroscience.2015.07.048. Farez, M.F., Mascanfroni, I.D., Mendez-Huergo, S.P., Yeste, A., Murugaiyan, G., Garo, L.P., et al., 2015. Melatonin contributes to the seasonality of multiple sclerosis relapses. Cell 162:1338–1352. http://dx.doi.org/10.1016/j.cell.2015.08.025. Filer, D., Patisaul, H.B., Schug, T., Reif, D., Thayer, K., 2014. Test driving ToxCast: endocrine profiling for 1858 chemicals included in phase II. Curr. Opin. Pharmacol. 19, 145–152. Goldman, S.M., Kamel, F., Ross, G.W., Bhudhikanok, G.S., Hoppin, J.A., Korell, M., et al., 2012. Genetic modification of the association of paraquat and Parkinson's disease. Mov. Disord. 27:1652–1658. http://dx.doi.org/10.1002/mds.25216. Grandjean, P., Landrigan, P.J., 2006. Developmental neurotoxicity of industrial chemicals. Lancet 368, 2167–2178. Hagerman, R., Hagerman, P., 2013. Advances in clinical and molecular understanding of the FMR1 premutation and fragile X-associated tremor/ataxia syndrome. Lancet Neurol. 12:786–798. http://dx.doi.org/10.1016/S1474-4422(13)70125-X. Hagerman, R., Au, J., Hagerman, P., 2011. FMR1 premutation and full mutation molecular mechanisms related to autism. J. Neurodev. Disord. 3:211–224. http://dx.doi.org/10. 1007/s11689-011-9084-5. Hodge, H.C., Maynard, E.A., Downs, W.L., Coye Jr., R.D., Steadman, L.T., 1956. Chronic oral toxicity of ferric dimethyldithiocarbamate (ferbam) and zinc dimethyldithiocarbamate (ziram). J. Pharmacol. Exp. Ther. 118, 174–181.
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Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Elucidating environmental dimensions of neurological disorders and disease: Understanding new tools from federal chemical testing programs Jennifer McPartland a,⁎, Heather Dantzker b, Christopher Portier a a b
Environmental Defense Fund, 1875 Connecticut Ave. NW, Ste. 600, Washington, DC 20009, USA Dantzker Consulting, LLC, 2613 N. Harrison St., Arlington, VA 22207, USA
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• ToxCast/Tox21 screening data can support hypothesis-driven research. • Federal chemical screening is currently limited for identifying neurotoxicants. • Broader scientific engagement can strengthen chemical testing programs. • Use of ToxCast and Tox21 by the broader scientific community is encouraged.
a r t i c l e
i n f o
Article history: Received 25 November 2016 Received in revised form 1 March 2017 Accepted 3 March 2017 Available online xxxx Editor: Jay Gan
a b s t r a c t Background: Federal agencies are making significant investments to advance predictive approaches to evaluate chemical hazards and risks. Environmental Defense Fund (EDF) believes that engagement with the broader scientific community is critical to building and maintaining a strong biological foundation for these approaches. Objectives: On June 18–19, 2015, EDF organized a meeting to 1) foster a conversation between federal scientists advancing predictive approaches and environmental health researchers investigating environmental exposures and neurological outcomes, and 2) explore opportunities and challenges for the use of federal chemical high-throughput in vitro screening (HTS) data in hypothesis-driven research toward, ultimately, improved data for public health decision-making. Discussion: The meeting achieved its objectives. Government scientists showcased their chemical testing programs and vision for how emerging data may be used to meet agency missions. Environmental health researchers shared their experiences using federal HTS data, offered recommendations for strengthening federal HTS platforms, and expressed great interest in continued engagement with evolving federal chemical testing initiatives. Conclusions: The meeting provided an invaluable exchange between two scientific communities with a shared interest in protecting public health from harmful environmental exposures, but who have not sufficiently engaged with each other. Discussions identified opportunities and work ahead for the use of HTS data in hypothesis-driven research. Though the meeting focused on neurological outcomes, the purpose,
⁎ Corresponding author at: 1875 Connecticut Ave. NW, Ste. 600, Washington, DC 20009, USA. E-mail addresses: [email protected] (J. McPartland), [email protected] (H. Dantzker), [email protected] (C. Portier).
http://dx.doi.org/10.1016/j.scitotenv.2017.03.024 0048-9697/© 2017 Elsevier B.V. All rights reserved.
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objectives and experience of the meeting are broadly applicable. EDF strongly encourages more discourse and collaboration between federal and non-government scientists working to understand environmental influences on health outcomes. © 2017 Elsevier B.V. All rights reserved.
1. Introduction As part of a larger portfolio of federal research efforts to advance new approaches for the evaluation of chemical toxicity and risk, vast quantities of high-throughput in vitro screening (HTS) data continue to be generated by the U.S. Environmental Protection Agency's (EPA) ToxCast™ initiative (U.S. EPA, 2017a) and the Tox21 partnership led by the National Institute of Environmental Health Sciences (NIEHS)/National Toxicology Program (NTP) (Tice et al., 2013, U.S. EPA, 2017b). Though these and related federal chemical testing programs have made significant progress since their inception, more growth opportunities remain for these programs to reach their full potential of protecting public health and the environment. Three goals must be achieved to realize the promise of predictive toxicology approaches in public health decision-making: 1) development of a scientific foundation supporting their use; 2) scientific acceptance of a new testing and analysis paradigm; and 3) health-protective policies that encourage their use (McPartland et al., 2015). Achieving these goals will require contributions from the broader scientific research community, particularly those on the forefront of understanding the etiology and pathology of environmentally-influenced disease (NRC, 2007). Environmental Defense Fund (EDF), in cooperation with the U.S. EPA and NIEHS/NTP, brought federal ToxCast™ and Tox21 scientists together with environmental health researchers for a meeting entitled, Elucidating Environmental Dimensions of Neurological Disorders and Disease: Understanding New Tools from Federal Chemical Testing Programs (UC Davis Conference Center, June 18–19, 2015). The meeting's objectives included 1) fostering a conversation between government scientists and environmental health researchers on federal predictive chemical testing approaches and 2) exploring opportunities and challenges for the use of these data in hypothesis-driven research. Progress on these objectives will strengthen links between researchers and regulatory scientists, improve the use of predictive toxicology data in decision-making, and help protect public health. Neurological outcomes affect millions of people worldwide: in the U.S. alone, approximately 60,000 people are diagnosed with Parkinson's disease each year (Parkinson's Disease Foundation, 2017); the percentage of children diagnosed with attention-deficit/hyperactivity disorder (ADHD) continues to rise from 7.8% in 2003 to 11.0% in 2011 (Visser et al., 2014); and in 2012, one in 68 children were estimated to have autism spectrum disorder (ASD), a 29% increase from 2008 (Christensen et al., 2016). Since genetic factors alone have been estimated to explain only 30 to 40% of all cases of neurodevelopmental disorders (Grandjean and Landrigan, 2006), understanding the role of the environment is critical if we are to better protect public health. Elucidating the impact of environmental exposures on neurological outcomes is particularly difficult owing to the underlying biological complexity of the nervous system. Approaches like HTS hold the potential to make progress toward identifying chemical exposures that interfere with normal brain development and function.
2016a, 2017c). The meeting then focused on how environmental health researchers are already integrating these data and tools for research on neurodevelopmental (e.g., autism) and neurodegenerative (e.g., Parkinson's) outcomes. Other presenters spoke to future possibilities for these data in basic and epidemiological research. Meeting participants were given the opportunity to engage with agency scientists on the various tools available to access and query federal HTS data. Throughout the meeting, participants exchanged ideas on key opportunities and challenges related to the use of HTS data in the field of environmental health. 2. Meeting outcomes Meeting outcomes include discussions on 1) recent experiences using ToxCast™ and Tox21 data in hypothesis-driven research investigations; 2) potential applications of HTS data in future hypothesis-driven research; and 3) current barriers to the use of federal HTS data by the broader scientific community. We then discuss measures that agencies and environmental health researchers can both take to further the conversation. 2.1. ToxCast™ and Tox21 data in hypothesis-driven research: recent investigations Compared to traditional toxicity testing approaches, HTS approaches are typically faster and provide greater insight into the mechanisms by which chemicals interfere with normal biology. These efficiencies provide researchers with tools and data to screen and identify chemical targets for additional investigation. HTS data can also be combined with other data types (e.g., ‘omics’, in silico models, whole animal laboratory studies, and epidemiological data) to enrich investigations of environmental impacts on health.
1.1. Overall meeting structure
2.1.1. Identifying novel targets for vitamin D receptor signaling Dr. Seth Kullman (Department of Biological Sciences at North Carolina State University) is using data from Tox21 to examine linkages between environmental exposures, modulation of vitamin D receptor (VDR) signaling, and later life neurodevelopmental consequences. Dr. Kullman shared that vitamin D deficiency early in development can lead to neurobehavioral outcomes through, among other proposed mechanisms, interference with dopaminergic signaling (Cui et al., 2015). Using data from an 8500-compound library screened through a Tox21 human VDR assay, Kullman's team identified multiple putative VDR agonists and antagonists. Orthogonal in vitro testing (e.g., human VDR transient transactivation assays and receptor-coregulator protein interactions assays) yielded generally concordant results. The strongest agonist and antagonist candidates were further examined for interaction with zebrafish VDR and demonstrated 100% concordance with the human VDR assays. Initial whole zebrafish studies of cadmium chloride, one of the identified VDR antagonists, revealed effects on larval locomotor activity. Dr. Kullman concluded that federal HTS data have been instrumental in identifying novel targets for his team's receptor of interest (VDR) that otherwise would have not likely been discovered.
The two-day meeting opened with presentations describing the ToxCast™ and Tox21 initiatives, including descriptions of their respective assay batteries, data, and accompanying user interfaces (U.S. EPA,
2.1.2. Using HTS data to develop prediction models of chemical toxicity Dr. Heather Patisaul (Department of Biological Sciences at North Carolina State University) investigates neuroendocrine disruption
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of brain regions and behavior, including those associated with ASD. Dr. Patisaul and colleagues set out to develop a prediction model for neurodevelopmental effects using ToxCast™ HTS data. Searching the ToxCast™ assay battery for steroid hormone and neuropeptide relevant assays, the research team identified assays for vasopressin (2 receptor binding assays) and oxytocin (1 receptor binding assay). Of the 1000 + chemicals assessed in these assays, Dr. Patisaul's team found 20 positive hits, including bisphenol-A (BPA). BPA has been shown to alter oxytocin, vasopressin, and dopaminergic neuron numbers in vivo and to decrease sociality in various animal models including male prairie voles, a species noted for its prosocial behaviors (Adewale et al., 2011; Sullivan et al., 2014; Wolstenholme et al., 2011, 2013). Dr. Patisaul and colleagues determined that too few ToxCast™ assays were available to develop a robust prediction model for neurodevelopmental toxicity and turned their efforts to the development of a prediction model for endocrine activity (Filer et al., 2014). One component of their model evaluates xenobiotic interference with monoamine signaling using 13 cell-free binding assays in ToxCast. Of the 1858 chemicals evaluated only chlorpromazine hydrochloride produced appreciable activity in this component of the model, suggesting a need for additional assays that examine chemical interactions with monoamine receptors. 2.1.3. Using HTS data for relational analysis and forecasting toxicity Dr. Scott Auerbach (Biomolecular Screening Branch, National Toxicology Program, NIEHS) uses federal HTS data to prioritize chemicals for further targeted investigation. Dr. Auerbach described a multi-step, integrated bioactivity profiling approach using publically-available data mining and visualization tools (NTP, 2017a). His approach operates on the premise that chemicals that exhibit similar biological activity across HTS assays will exhibit similar in vivo biological/toxicological properties. As an illustration, Dr. Auerbach used triethyltin, a well-documented neurotoxic compound, to identify other potential neurotoxic chemicals in the Tox21 chemical library. Zinc dimethyldithiocarbamate and zinc pyrithione were identified. Additional cursory review of the literature revealed that zinc dimethyldithiocarbamate has been shown to cause motor changes when administered to rats (Hodge et al., 1956), as well as an association with Parkinson's disease (Moretto and Colosio, 2013), and that zinc pyrithione has resulted in toxicity to the peripheral nerve tissue and skeletal muscle in lab animals (SCCS, 2014). Dr. Auerbach also illustrated a “crowd-sourcing” approach that involves using chemical-to-biological annotations of nearest neighbors as ascribed in various databases (e.g., DrugBank, Toxin Target, Leadscope, PubMed MeSH terms etc.). Using zinc pyrithione as a hypothetical chemical with undefined toxicity, applying this approach yielded several biological annotations related to developmental toxicity, a finding consistent with existing data showing effects on neuronal signaling that may be related to observed paralytic effects (SCCS, 2014). 2.1.4. Using HTS data to complement zebrafish investigations of chemical toxicity Dr. Robert Tanguay (Department of Molecular and Environmental Toxicology at Oregon State University) described his efforts to identify and evaluate chemical effects on embryonic development in zebrafish (i.e., between 6 and 120 h post fertilization). Dr. Tanguay and collaborators blindly evaluated 1060 ToxCast™ chemicals in: 1) an embryonic photomotor response assay that examines changes in a well-defined embryonic photomotor response to high-intensity light (Reif et al., 2016); 2) a larval locomotor response assay that measures changes in swimming behavior during alternating periods of dark and light; and 3) an assay for aberrations in 22 distinct morphological endpoints (e.g., notochord, yolk sac) (Truong et al., 2014). They found 376 active compounds in the embryonic photomotor response assay, many exhibiting non-monotonic dose-response curves.
Embryonic photomotor response appeared to be predictive of morphological teratogenic outcomes observed four days later. Dr. Tanguay emphasized that HT in vivo assays (e.g., zebrafish) can provide rapid data for chemical prioritization, structure-based prediction models, and phenotypic anchoring for adverse outcome pathway discovery. Dr. Francisco Quintana (Department of Neurology at Brigham and Women's Hospital and Harvard Medical School) is interested in understanding how environmental influences may contribute to inflammation-induced neurodegeneration in multiple sclerosis (MS). Dr. Quintana focuses on immune system drivers of progressive loss of neuronal myelin in both relapsing-remitting MS—driven largely by abnormal immune activity by effector (pathogenic) T cells—as well as secondary progressive MS, driven largely by astrocytes (Farez et al., 2015, Mayo et al., 2014, Rothhammer et al., 2016). Dr. Quintana and collaborators used ToxCast™ data measuring bioactivity against biological targets associated with autoimmune disease pathways (i.e., AHR, TNF, IFN, STAT3, TGFb1, IL1a, JAK2) to select compounds for screening in a zebrafish model of T-cell dependent inflammatory bowel disease (IBD) (McGovern et al., 2015). He explained that T cells that cause pathology in IBD share many features with T cells that cause pathology in MS (Marson et al., 2015). Eighty-five ToxCast™ compounds of interest were screened in the zebrafish model of colitis with 24 compounds showing significant inflammatory or anti-inflammatory effects. Four of these (2 inflammatory and 2 anti-inflammatory) have been selected for additional studies in mice. 2.2. ToxCast™ and Tox21 data in hypothesis-driven research: Potential future applications Looking forward, meeting participants explored how federal HTS data may be used in future research. 2.2.1. Using federal HTS data to inform epidemiological investigations Dr. Newschaffer (AJ Drexel Autism Institute, Drexel University) described how epidemiological approaches could employ large HTS data sets to 1) identify additional ASD exposure candidates and 2) help create candidate ASD ‘subexposomes’ from large exposomic data sets. He explained that HTS data could broaden the scope of candidate chemicals for epidemiological research and that perturbations of biological pathways identified through HTS could be interpreted as signals to be measured in human biological samples. One such opportunity described by Dr. Newschaffer is in Genome-Environment Wide Interaction Studies (GEWIS), whereby chemical HTS data could be used to identify exposures to measure and assess. As an example, he highlighted the ongoing Study to Explore Early Development (SEED) case-control investigation—currently one of the largest studies in the U.S. to identify environmental and other factors that put children at risk for ASD (Schendel et al., 2012). Dr. Tanner (Department of Neurology, University of California, San Francisco, and Parkinson's Disease Research, Education, and Clinical Center, San Francisco Veterans Affairs Health Care System) provided several examples of how HTS data could be useful for PD environmental epidemiology. The street drug, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes PD-like symptoms in people (Calne and Langston, 1983). Mining HTS data for compounds with similar mechanistic characteristics as MPTP would help identify other potential environmental contributors to PD. For compounds identified in human studies of PD (e.g., paraquat and 2,4-D), such a database could provide additional insight into their mechanisms of action considering what is known and unknown about PD etiology and pathogenesis. As a final example, Dr. Tanner highlighted that in her earlier investigations of farmworkers and PD (Tanner et al., 2011), access to HTS mechanistic data would have facilitated consideration of a broader set of compounds to investigate beyond the pesticides she studied. Because gene–environment interaction appears to be important to the etiology of PD (Goldman et al., 2012), databases integrating
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genetic information and pathways for xenobiotic metabolism would be especially helpful. 2.2.2. Research approaches using HTS data to address neurological health outcomes Meeting participants outlined several research proposals for how HTS data can be integrated into hypothesis-driven research related to neurological disease outcomes, and specifically how federal HTS assay batteries could be further augmented toward this end. One proposal was to mine federal HTS datasets to identify chemicals that may impact attention, specifically ADHD, by searching for chemicals structurally and biologically (i.e., shared bioactivity in the HTS assays) similar to polybrominated diphenyl ethers (PBDEs)—a chemical class associated with neurological effects including effects on attention (Berghuis et al., 2015, Cowell et al., 2015, Sagiv et al., 2015). Identified compounds would be assessed in zebrafish for methylomic and transcriptomic effects resulting from embryonic exposures to select an even smaller subset for testing in mammalian models of attention, and eventually epidemiological studies, assuming biomarkers for these compounds exist or could be developed. A similar approach was highlighted as potentially useful for investigations of PD. Here however, more emphasis was placed on mining the federal HTS data for chemicals with shared bioactivity profiles since canonical, structurally-similar compounds associated with PD, like paraquat and MPTP, operate through very different mechanisms. Again, promising compounds would be further examined in animal and ultimately epidemiological studies. Another proposal focused on broadening the NTP's S1500 transcriptomic project, part of the larger Tox21 initiative, that seeks to measure chemically-induced changes in gene expression across a sentinel set of 1500 environmentally-responsive genes (NTP, 2017b) to identify environmental exposures related to autism. Specifically, full transcriptomic sequencing in relevant neurons could be done and compared to extant autism gene databases such as the SFARI autism gene list (SFARI, 2017). Others suggested using federal HTS data to interpret metabolomics data from an autism case-control study on cord blood in a large, previously well-characterized cohort. A final research proposal focused on neurodegenerative disorders began with the identification of compounds in the current federal HTS libraries more likely to reach the brain based on hydrophilicity and hydrophobicity. In parallel, a set of genes relevant to neurodegenerative disease would be compiled and correlated to targets currently represented in the federal HTS assays using large, highly-curated genomic databases such as NextBio (NextBio, 2017). Data from the correlated assays would be cross-referenced against compounds identified as likely to cross the blood-brain barrier to identify a targeted set of chemicals for additional assessment. It is important to note that few of the canonical genes relevant to late onset neurodegenerative outcomes are currently represented in the federal HTS testing platforms but such assays could be developed. 2.3. Critical biological realities to consider in advancing predictive chemical testing Various, inherent biological complexities continue to challenge the development of HTS platforms for neurodevelopmental and neurodegenerative outcomes, but significant progress is being made. These include accounting for influencing risk factors such as genetic and gender susceptibilities and timing of exposure, and integrating metabolic competence into HTS assays. 2.3.1. Neuro-relevant biological targets Some assays for assessing neurological endpoints are included in the federal HTS platforms but more are needed, including: • Assays that examine pharmacological manipulation of neurotransmission through, for example, receptors for oxytocin, serotonin,
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GABA, vasopressin, glutamate, and dopamine; • inclusion of assays that interrogate pathways involved in shaping neuronal connectivity in the developing brain (e.g., dendritic morphogenesis, synapse formation, and synapse stabilization); • greater representation of ASD sentinel genes, and • greater representation of genes and pathways associated with PD including α-synuclein aggregation (e.g., PARK I, PARK IV and LRRK2), degradative pathways (e.g., proteasomal degradation and autophagy), and mitochondrial dysfunction. 2.3.2. Differential genetic susceptibility Genetic susceptibility, and other forms of individual susceptibility, are critical considerations in the evaluation of potential health effects resulting from environmental exposures. However, comprehensively accounting for these susceptibilities has proven challenging for chemical testing broadly. Dr. Pamela Lein (Department of Molecular Biosciences at the University of California, Davis) described how in vitro testing may help to obtain “first-pass” information on differential effects of chemicals on different genetic backgrounds. One approach she is developing in collaboration with Dr. Isaac Pessah (Department of Molecular Biosciences, University of California, Davis) involves evaluating in vitro chemical exposures on neurotypical neurons versus neurons expressing ASD-associated gene variants like the Fragile X premutation (FMR1 containing CGG trinucleotide repeats). The Fragile X premutation is the single most prevalent gene disorder associated with ASD (Hagerman et al., 2011; Hagerman and Hagerman, 2013; Krueger and Bear, 2011; Leehey and Hagerman, 2012). Since the FMR1 gene is X-linked, two cell subpopulations, neurotypical and FMR1 premutation, can be isolated from the same human iPSC-derived neuronal cells for in vitro testing (Liu et al., 2012). Already her collaborator, Dr. Pessah, has shown differences in calcium signaling—important in determining the extent of dendritic arborization—between these two cell sub-populations. The plan is to now screen compounds in these two neuronal subpopulations for differential effects on neuronal connectivity to better understand how specific gene-environment interactions can render some individuals more susceptible to neurodevelopmental disorders from the same environmental exposures. Dr. Valerie Hu (Department of Biochemistry and Molecular Medicine at The George Washington University) described her efforts to elucidate the sexual dimorphism of ASD, and how this has led to the development of a new HTS assay to screen for environmental contributors to ASD. Her research focuses on retinoic acid-related orphan receptor alpha (RORA). RORA is an ASD target of interest identified from large-scale microarray analyses of autism candidate genes (Nguyen et al., 2010). Dr. Hu's team has found that RORA transcriptionally regulates over 2500 potential targets, including over 400 autism risk genes (Sarachana and Hu, 2013). Moreover, previous animal studies examining RORA deficiency have revealed functional deficits and sexually dimorphic effects in line with ASD pathology and pathophysiology. Because of the hormonal sensitivity of RORA (Sarachana et al., 2011), Dr. Hu and her team are in the process of developing a high-throughput luciferase screening assay to identify endocrine disrupting chemicals that influence RORA expression. 2.3.3. Timing of exposure Timing of exposure is another critical consideration in the evaluation of potential health effects resulting from environmental exposures. However, accurately accounting for the timing of exposure can present challenges for in vitro chemical testing, particularly when evaluating complex endpoints like neurodevelopmental toxicity which involves dynamic, temporally and spatially orchestrated biological processes. Dr. Elaine Faustman (Department of Environmental and Occupational Health Sciences at the University of Washington) presented research designed to temporally anchor in vitro cellular models of development to in vivo models of development. One approach Dr. Faustman's team is pursuing uses human neuroprogenitor stem cells
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(hNPCs) to examine forebrain development, specifically formation of cortical pyramidal neurons. Dr. Faustman is using the hNPC model to identify and define transcriptomic profiles in vitro that correspond to biological activity of humans at 5 to 10 weeks of gestation. The transcriptomic profiles of hNPC cells are tracked over 21 days in vitro. Comparison of gene expression in undifferentiated hNPC (day 0) versus the fetal brain samples shows a significant degree of concordance between the in vitro and in vivo transcriptomes (Wegner et al., 2017 in press). Genes differentially expressed across time both in vitro and in vivo were enriched for gene ontology (GO) terms associated with developmental processes including brain development. Genes with differential expression uniquely in vitro were associated with GO terms related to stress signaling, and genes with differential expression uniquely in vivo were associated with GO terms related to neurotransmission. Dr. Faustman indicated that these findings were not surprising given that there is some degree of cellular stress resulting merely from growth in culture (e.g., artificial air environment) and the well-established importance of cell-cell contact occurring in vivo among specific cell populations during neurodevelopment (Wegner et al., 2017 in press). Dr. Faustman suggested that this type of exploratory, anchoring data provides critical information for interpreting HTS data in terms of understanding what aspects of brain development are being captured in a particular in vitro testing model and which are not. It may be that a chemical acts on a neurodevelopmental pathway that is active during a specific time period of brain development and if an in vitro test system does not reflect the biology occurring during that time period then the chemical effect can be missed. 2.3.4. Metabolic competence and other issues common to HTS platforms Metabolic competence (i.e., accounting for the generation of metabolites in toxicity testing), the inherent artificial conditions of in vitro testing, as well as the unique biology of neuroprogenitor cells and neurons that may be absent in the cell lines typically used in HTS platforms all represent challenges for HTS broadly. Agency scientists are at the forefront of tackling these types of challenges for the broader research community. For example, federal agencies collaborated to launch a crowdsourcing effort, “Transform Tox Testing Challenge: Innovating for Metabolism” to identify solutions to better integrate biotransformation into its HTS assays (U.S. EPA, 2016b)—an investment that benefits all those endeavoring to employ HTS in their investigations. 2.4. Opportunities for the use of HTS data by the broader scientific community 2.4.1. Access and use of HTS data and tools Genius Bar sessions at the meeting provided participants with the opportunity to explore web-based federal HTS data dashboards and related query tools (U.S. EPA, 2016a, 2017a, 2017c) alongside expert EPA and NIEHS scientists involved in their development and application. These sessions were widely regarded as one of the best features of the meeting. Participants cited the value and need for more user guidance and tutorials; additional clarity around data processing decision rules and hit calls; and other information to help researchers interpret query results and conduct further customized analyses. Communication of when updates, corrections, or additions are made to these web-based data interfaces will continue to be an important factor in increasing use of these valuable resources. 2.4.2. Opportunities for building a user community Growing an active community of federal HTS data users, which would evolve over time into a peer-support network, would benefit both research scientists as well as resource-constrained agencies. Researchers and agency scientists would mutually benefit from dedicated agency staff who could field inquiries from external users related to the emerging data, tools, and analyses. Meeting participants expressed enthusiasm for these types of activities.
Such a user community would also benefit from hands-on, interactive training opportunities. One possible approach would be to incorporate coursework on the federal HTS data and dashboards into collegiate and graduate academic programs. Training via workshops, either regional, traveling, or those in which participants would be funded to travel would also be highly valuable and desired training opportunities. 3. Discussion In the Davis meeting, EDF met its near-term objectives of 1) fostering a conversation between government scientists and environmental health researchers to strengthen emerging predictive chemical testing approaches; and 2) exploring opportunities and challenges for the use of resultant data in hypothesis-driven research. Participants examined some of the research opportunities that can be gained by introducing environmental health scientists to the ToxCast™ and Tox21 initiatives. This proved to be a win-win situation for both the researchers and the regulatory agencies. Environmental health researchers left the meeting with a much stronger understanding of federal HTS programs and how to access and utilize federal HTS data for hypothesis-driven research. Many also left with knowledge of new compounds that may be relevant to their research on neurodevelopmental and neurodegenerative disease. Over time, as these researchers gain greater understanding of disease etiology and pathogenesis, more will be known about which cellular targets are related to certain diseases, providing regulatory agencies with new knowledge that can be integrated into chemical testing platforms. While this meeting focused on neurological outcomes, it is our hope that new conversations and collaborations will emerge between agency scientists and environmental health researchers working in other disease areas. Neurodevelopment and neurodegeneration are complex, multifaceted processes that are not fully understood. The datasets being created for regulatory chemical screening have great value beyond their original purpose. As Drs. Kullman, Patisaul, Hu, and others described, federal HTS data have the potential to help basic researchers who are examining the effect of chemicals on biological processes to further illuminate how those processes relate to disease. The datasets can also provide new chemical targets for epidemiological investigations of threats to health from environmental exposures, such as described by Drs. Newschaffer and Tanner. Examining very complicated processes with multiple targets, as described by Dr. Lein, Tanguay, Auerbach and others, benefits greatly from having large, searchable chemical databases. Potential interactions of multiple chemicals altering multiple targets in a process can be hypothesized through such a database then further examined through complementary experimentation. Currently, however, ToxCast™ and Tox21 are neither comprehensive in chemical nor biological space. We encourage agency scientists to continue to be transparent about limitations in the current HTS platforms so that external scientists can assess the utility of the data and, importantly, identify opportunities to fill data gaps. A key way for investigators to help address gaps in the datasets is to learn about the screening efforts, query the HTS data to identify what is useful to them in their own hypothesis-driven research, and simultaneously, identify what may be missing in the current assay batteries. Researchers can nominate additional assays for inclusion in federal chemical screening programs using the Tox21 assay nomination process (U.S. EPA, 2017d). In parallel, we also encourage the Tox21 and ToxCast™ programs to continue to engage with basic researchers regarding the types of assays that would be most useful for identifying disease threats, as was done by the National Toxicology Program in a 2012 workshop and subsequent work on diabetes and obesity (Thayer et al., 2012, Auerbach et al., 2016). These simultaneous activities will broaden the universe of scientists collectively working to advance and accelerate federal HTS and similar efforts. The overarching take-away from the meeting is that, despite certain challenges, federal HTS data present basic researchers and
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epidemiologists with a rich opportunity for exploration, inquiry, and collaboration, which in turn presents agencies with a mechanism for advancing its chemical testing programs. By so doing, agencies can fill current gaps, resulting in better coverage of disease-relevant processes in these test systems, and, ultimately, contributing to a more robust understanding of environmental influences on disease. Competing financial interests Dr. Heather Dantzker works as a consultant to the Environmental Defense Fund. Dr. Jennifer McPartland and Dr. Christopher Portier declare they have no actual or potential competing financial interests. Acknowledgements The authors would like to thank each of the participants and presenters at EDF's June 2015 Elucidating Environmental Dimensions of Neurological Disorders and Disease: Understanding New Tools from Federal Chemical Testing Programs meeting, including the U.S. EPA and NIEHS/ NTP staff who attended and supported the meeting's Genius Bar sessions. In addition, we would like to especially thank each of the presenters who allowed us to highlight their research in this paper and provided timely review of drafts. Finally, we would like to thank Autism Speaks and the National Parkinson Foundation for providing participant travel support to the meeting and the National Toxicology Program for issuing a small number of competitive travel awards. References Adewale, H.B., Todd, K.L., Mickens, J.A., Patisaul, H.B., 2011. The impact of neonatal bisphenol-A exposure on sexually dimorphic hypothalamic nuclei in the female rat. Neurotoxicology 32:38–49. http://dx.doi.org/10.1016/j.neuro.2010.07.008. Auerbach, S., Filer, D., Reif, D., Walker, V., Holloway, A.C., Schlezinger, J., Srinivasan, S., Svoboda, D., Judson, R., Bucher, J.R., Thayer, K.A., 2016. Prioritizing environmental chemicals for obesity and diabetes outcomes research: a screening approach using ToxCast™ high-throughput data. Environ. Health Perspect. 124:1141–1154. http:// dx.doi.org/10.1289/ehp.1510456. Berghuis, S.A., Bos, A.F., Sauer, P.J., Roze, E., 2015. Developmental neurotoxicity of persistent organic pollutants: an update on childhood outcome. Arch. Toxicol. 89:687–709. http://dx.doi.org/10.1007/s00204-015-1463-3. Calne, D.B., Langston, J.W., 1983. Aetiology of Parkinson's disease. Lancet 2, 1457–1459. Christensen, D.L., Baio, J., Braun, K.V., Bilder, D., Charles, J., Constantino, J.N., et al., 2016. Prevalence and Characteristics of Autism Spectrum Disorder Among Children Aged 8 years — Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States, 2012. MMWR Surveill Summ 65:pp. 1–23. http://dx.doi.org/10.15585/ mmwr.ss6503a1 Available at: http://www.cdc.gov/mmwr/volumes/65/ss/ss6503a1. htm (Accessed 28 Feb 2017). Cowell, W.J., Lederman, S.A., Sjödin, A., Jones, R., Wang, S., Perera, F.P., et al., 2015. Prenatal exposure to polybrominated diphenyl ethers and child attention problems at 3–7 years. Neurotoxicol. Teratol. 52 (PtB):143–150. http://dx.doi.org/10.1016/j.ntt.2015. 08.009. Cui, X., Pertile, R., Liu, P., Eyles, D.W., 2015. Vitamin D regulates tyrosine hydroxylase expression: N-cadherin a possible mediator. Neuroscience 304:90–100. http://dx.doi. org/10.1016/j.neuroscience.2015.07.048. Farez, M.F., Mascanfroni, I.D., Mendez-Huergo, S.P., Yeste, A., Murugaiyan, G., Garo, L.P., et al., 2015. Melatonin contributes to the seasonality of multiple sclerosis relapses. Cell 162:1338–1352. http://dx.doi.org/10.1016/j.cell.2015.08.025. Filer, D., Patisaul, H.B., Schug, T., Reif, D., Thayer, K., 2014. Test driving ToxCast: endocrine profiling for 1858 chemicals included in phase II. Curr. Opin. Pharmacol. 19, 145–152. Goldman, S.M., Kamel, F., Ross, G.W., Bhudhikanok, G.S., Hoppin, J.A., Korell, M., et al., 2012. Genetic modification of the association of paraquat and Parkinson's disease. Mov. Disord. 27:1652–1658. http://dx.doi.org/10.1002/mds.25216. Grandjean, P., Landrigan, P.J., 2006. Developmental neurotoxicity of industrial chemicals. Lancet 368, 2167–2178. Hagerman, R., Hagerman, P., 2013. Advances in clinical and molecular understanding of the FMR1 premutation and fragile X-associated tremor/ataxia syndrome. Lancet Neurol. 12:786–798. http://dx.doi.org/10.1016/S1474-4422(13)70125-X. Hagerman, R., Au, J., Hagerman, P., 2011. FMR1 premutation and full mutation molecular mechanisms related to autism. J. Neurodev. Disord. 3:211–224. http://dx.doi.org/10. 1007/s11689-011-9084-5. Hodge, H.C., Maynard, E.A., Downs, W.L., Coye Jr., R.D., Steadman, L.T., 1956. Chronic oral toxicity of ferric dimethyldithiocarbamate (ferbam) and zinc dimethyldithiocarbamate (ziram). J. Pharmacol. Exp. Ther. 118, 174–181.
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