David Reif

Background: The chemicals are diverse. They contain a lot of fluorine molecules. Elemental substitutions can change their properties. Different structural moieties (carbolic acid, sulfonic acid, ethers, etc.) change their properties and influence where they go in the environment, whether and how organisms take them in, how they distribute within the organism, and how they influence various biological targets. Objective: Conduct the largest comprehensive in vivo, structure-activity based toxicity studies of per- and polyfluoroalkyl substances (PFAs) and understand the partitioning of PFAs. Experimental approach: We will procure and build the largest PFA library and expose embryonic zebrafish to all PFAs in a wide range of concentrations to investigate toxicity. For each concentration of each chemical, the body burden of PFAs in zebrafish embryos as a function of time (1, 2 and 5 days) to determine its ability to enter the embryo, prior to metabolism and after an active metabolism has been established. The ability of PFAs to enter the embryo maybe impacted based on whether they will stay in the water, or stick on various surfaces. To account for this, we propose to conduct studies to determine the partitioning of the chemical structure for a subset of the PFAs at various concentrations and model the behavior of the chemical in water. The data collected in the zebrafish model will prioritize PFAs to be the most toxic and assessed in mice during early developmental stage and measure adaptive and innate immune responses as well as splenic and thymic immunophenotype. Organ samples will be taken from mice exposed to various PFAs and the concentration of PFAs will be measured. The measurements will be used to build a pharmacokinetic model that explains and predicts concentrations of PFAs in various organs of mice as a function of exposure dose. In parallel, adult zebrafish will be exposed to PFAs through dietary ingestion and the concentration of PFAs in various organs and the water will be measured over time. This data will help build a pharmacokinetic model. Expected outcome: The toxicity results from the embryonic zebrafish and mice, and the pharmacokinetic models will help EPA prioritize chemicals for further testing and may also alert chemical manufacturers that some of their commercial products may be toxic. The proposed work will help establish a knowledge base that can help predict chemical toxicity without in vivo testing.

The PVI is a collaborative effort led from NIEHS and NCSU. This project will include updating the modeling, maintaining the web tools, and developing stand-alone software for others to implement their own similar modeling.

Our proposed project will seek to explore the relationship between greenspace exposure and telomere length in a large sample from the United States. This result can provide evidence for a biological pathway that greenspace exposure influences human health. The project will also examine the spatial scale of the exposure relationship to determine the feasibility of Zip codes as an analysis unit versus census geographies and residential location. If Zip codes provide sufficient results, this spatial information would provide a means of data collection in future studies that protects participant privacy. The proposed project will expand CHHE collaborations with new connections between Drs. Hipp and Reif. Importantly, pilot funding will also provide Dr. Ogletree, postdoctoral scholar in Parks, Recreation, and Tourism Management and the Center for Geospatial Analytics, with valuable experience to establish himself in the field of environmental health research and with NHANES data, expanding the potential for a K award.

The overall goal of this research proposal is to gain a molecular understanding of the interactions between engineered nanomaterials (ENM) and the embryonic zebrafish. To advance our understanding and keep pace with new innovations, we increasingly need advances in ENM synthesis, biological screens and sustainability metrics. Due to the complex nature of their interactions, these areas must be addressed in the context of a highly interactive multi-disciplinary team. We propose to achieve the following research objectives: 1. Obtain multifunction ENMs with precisely defined structures to address key questions regarding how molecular level structure influences toxicity. 2. Advance the comprehensive, rapid, multi-dimensional in vivo screening approaches needed to inform design guidelines to reduce the adverse impacts associated with ENMs. 3. Collaborate with other U01 consortia investigators to develop principles that may be used to design new nanoparticles with reduced environmental impacts. We will initially collaborate with the Nanomaterials Health Implications Research (NHIR): Engineered Nanomaterials Resource and Coordination Core (ERCC). We expect that this core will provide precision-engineered, multifunction ENMs to probe the roles of surface chemistry and core characteristics in function and properties. We will rapidly evaluate bioimpacts of the reference materials using a multi-dimensional, high throughput embryonic zebrafish bioassay that we have pioneered

The graduate training program in Bioinformatics at North Carolina State University requests support for 10 pre-doctoral trainees and 2 post-doctoral trainees. These trainees work at the interface of genomic science, computer science and statistics.

The central hypotheses of this proposal are that: (i) stem cell-derived cardiomyocyte cultures constitute an effective organotypic culture model for predictive toxicity screening of environmental chemicals; (ii) a population-based experimental design utilizing a panel of human iPSCs and mouse Collaborative Cross (CC) can assess variation in toxicity to better characterize uncertainties; and (iii) integration of dosimetry with screening provides an in vivo context to in vitro data and improves human health assessments. Project 1 will conduct population-based concentration-response high-content/-throughput in vitro screening of up to 200 ToxCast chemicals in iPSC-derived cardiomyocytes from 100 humans and collect pharmacokinetic data using hepatocytes. Project 2 will conduct mouse population-based in vitro screening of these chemicals in CC ESC-derived cardiomyocytes followed by in vivo validation in the CC strains. Project 3 will conduct dose-response modeling to establish appropriate point of departure, genome-wide association analyses and in vitro-to-in vivo extrapolation modeling.

Exposure to environmental chemicals has been linked to increases in cancer incidence, birth defects, impaired cognitive development, and neurodegenerative disease. Unfortunately, the gap between the ever-expanding number of chemicals in the environment and data on their potential health hazards continues to widen. Although recent advancements that use in vitro, high-throughput screening (HTS) technologies may speed the pace of chemical testing, those platforms cannot detect adverse health effects diagnosable only at a systemic level, such as abnormal development or aberrant behavior. Additionally, an in vivo context is needed to quantify the contribution of interindividual genetic variation to susceptibility differences in developmental or behavioral consequences of exposure. There is strong evidence that gene-environment interactions (GxE) related to individual genetic variation play an important role in health outcomes, and that these interactions are likely a major source of the heterogeneity in response to chemical exposure. Thus, understanding the role of GxE in differential susceptibility to chemical exposure will be key to protecting public health. We propose development of a collaborative bioinformatic + experimental system to study health outcomes affected by gene-environment interactions (Y=GxE) that comprehensively describes (Y), refines characterization of (E), then investigates and probes (G). This system will leverage massive data generated by HTS of chemicals through morphological and behavioral assays in embryonic zebrafish during the critical period (the first 5 days immediately after fertilization) when developmental processes are most highly-conserved between this vertebrate model organism and humans. These data will be analyzed to quantify GxE that elicit differential health outcomes following chemical exposure. The lasting significance of this proposal will be a scalable, efficient system to rapidly address questions of differential genetic susceptibility to an expanding chemical universe.

The mission of the Center for Human Health and the Environment (CHHE) is to advance understanding of environmental impacts on human health. Through a systems biology framework integrating all levels of biological organization, CHHE aims to elucidate the fundamental mechanisms through which environmental exposures/stressors interface with biomolecules, pathways, the genome, and epigenome to influence human disease. CHHE will develop three interdisciplinary research teams that represent NC State’s distinctive strengths. CHHE will implement specific mechanisms to promote intra- and inter-team interactions and build interdisciplinary bridges to advance basic science discovery and translational research in environmental health science along the continuum from genes to population. These teams are; - The Molecular/Cellular-Based Systems and Model Organisms Team will utilize cutting edge molecular/cellular-based systems and powerful vertebrate and invertebrate model organisms to define mechanisms, pathways, GxE interactions, and individual susceptibility to environmental agents. - The Human Population Science Team will integrate expertise on environmental exposures, epidemiology, genomics and epigenomics to identify key human pathways and link exposure and disease across populations. - Bioinformatics Team will develop novel analytics and computational tools to translate Big Data generated across high-throughput and multiscale experiments into systems-level discoveries To further increase the impact and translational capacity of these teams, CHHE will develop three new facility cores that will provide instrumentation, expertise, and training to facilitate basic mechanism- to population-based research. - The Integrative Health Sciences Facility Core will expand the ability of CHHE members to translate basic science discoveries across species and provide mechanistic insights into epidemiological studies by partnering with: a) NC State’s Comparative Toxicogenomics Database (CTD); b) East Carolina University Brody School of Medicine and c) NC Dept. of Health and Human Services. - The Comparative Pathobiology Core will be located at NC State’s top-ranked College of Veterinary Medicine and its nationally recognized veterinary pathology group to facilitate assessment of the effects of environmental stressors in the many model organisms utilized by CHHE members. - The Systems Technologies Core will introduce state-of-the-art proteomics capabilities and dedicated bioinformatics support to expand the ability of CHHE members to analyze the Next Generation Sequencing data involving the genome, transcriptome and epigenome. As a land-grant university, NC State has an extensive and active Cooperative Extension Service network throughout North Carolina. CHHE will utilize this unique network to develop a highly effective, multi-directional Community Outreach and Engagement Core to disseminate findings that will contribute to addressing disparity in exposures and health outcomes and to educate communities about environmental influences on health. A strong Career Development Core for early stage scientists that is coordinated with a robust Pilot Project Program will support cutting-edge, collaborative and multidisciplinary environmental health projects to enhance the research success and impact of our membership. Through these activities and the purposeful interfacing of different disciplines CHHE will build on NC State’s unique research and community outreach strengths to become a premier transformative and synergistic EHS Core Center.

In this project, Dr. Denis Fourches (Assistant Professor, Department of Chemistry, North Carolina State University) and Dr. David Reif (Associate Professor, Department of Biological Sciences, North Carolina State University) will consult with CalEPA’s Office of Environmental Health Hazard Assessment (OEHHA). Both Fourches and Reif are resident members of the Bioinformatics Research Center, which houses the graduate students, postdocs, and staff of their respective labs. The pair has a strong history of collaboration on projects that bring together expertise in Computational Toxicology, Risk Assessment, Cheminformatics, and Bioinformatics.

Objective: A team of researchers from Oregon State University and North Carolina State University proposes to conduct the first comprehensive in vivo toxicity studies of flame retardant chemicals (FRCs), including FRCs that EPA has banned, FRCs that companies manufacture now, and FRCs that companies have proposed as alternatives. We will test the hypothesis that the toxicity of FRCs will be highly dependent on their chemical structure. Experimental Approach: We will expose embryonic zebrafish to FRCs and observe their morphology and behavior for signs of toxicity. We will also grow exposed zebrafish to adulthood and observe their morphology and behavior for signs of toxicity. We plan to discover which FRCs have the lowest hazard potential and to rank the others according to their toxicity. With the identified phenotypic anchors, we will conduct whole-transcriptome analyses to define the early expression changes and pathways underlying the adverse outcomes to the toxic FRCs. in FRC outcomes across the levels of biological organization, i.e, chemical structure, similarity of gene expression profiles, early and adult life stage adverse outcomes, and thereby define adverse outcome pathways (AOP) for mechanistic FRC hazard prediction. We plan to bin FRCs according to their mode of action and to discover how an FRC’s mode of action depends on its structure, thereby gaining the ability to predict the toxicity of new compounds. We will develop new tools that enable manufacturers and risk assessors to determine the likelihood that a new compound is safe. Expected Outcome: The proposed toxicity screening results will help EPA prioritize chemicals for further testing and may also alert chemical manufacturers that some of their commercial products may be toxic. The identified AOPs will improve the research community’s ability to translate zebrafish results to other species. If high-throughput, low-cost zebrafish testing closely replicates the results of rodent testing, then zebrafish testing, in combination with other assays, may eliminate the need for rodent testing, at least for certain classes of chemicals. The proposed work will help to establish a base of knowledge that will lead to novel cell-based assays that can reliably predict chemical toxicity without in vivo testing.