Seth Kullman

The North Carolina State University ����������������Molecular Pathways to Pathogenesis in Toxicology��������������� NIEHS T32 training grant is a long-standing, impactful and multidisciplinary initiative that has supported 130 pre-doctoral trainees and 18 post-doctoral researchers over the past 41 years. Graduates of this program conduct basic and applied research, teach at universities and colleges, evaluate product safety, and assist public agencies and private industries in resolving important public health and environmental problems. Our mission is to provide the next generation of toxicologists/environmental health science (EHS) researchers with the technical, operational and professional skills necessary to conduct high impact EHS research, communicate effectively to a wide variety of audiences, and work as part of multidisciplinary teams to understand how human health is impacted by environmental factors. In this competitive renewal, we will continue with the current Molecular Pathways to Pathogenesis in Toxicology training theme and our overarching systems biology framework. This approach aims to integrate all levels of biological organization from biomolecules to human populations to elucidate the fundamental mechanisms through which environmental stressors influence human health outcomes. We have added 10 new research-active faculty for a total of 28 mentors, enhanced our mentor training requirements, reorganized key research areas, and updated core curricula. We have enhanced our core professional development series in scientific rigor, reasoning, experimental design and methods, and data analysis and interpretation through formal training in science communication, training in grant writing, coding in ����������������R���������������, and methods for enhancing reproducibility. Participating mentors of this NIEHS T32 training grant are supported by research grants from NIEHS, other NIH Institutes, other federal agencies including DOD, EPA, NSF, state agencies and private foundations. Environmental health science research at NC State has never been stronger as evidenced by the renewal of our NIEHS EHS Core Center (P30) and the recent award of a Superfund Research Program (SRP) (P42). These centers serve to facilitate collaborations among participating mentors/trainees and provide access to cutting-edge institutional infrastructure and financial support to advance EHS research at NC State. Both centers serve as an extraordinary resource for trainees through access to core facilities, sponsored symposia, seminars, workshops and professional development opportunities. The NIEHS T32 training program is the foundation of NC State������������������s highly ranked graduate program in toxicology; our well-qualified mentors, strong pool of applicants, and rigorous and comprehensive EHS training opportunities will ensure the continued success of this initiative. We request continued support for six trainees.

Mass spectrometry is an extraordinarily powerful bioanalytical technique that has had a profound impact on our molecular understanding of human health and disease. Major advances in mass analyzer technology, dissociation techniques, lasers, and ionization methods are largely attributed to the central role that mass spectrometry plays in the field of systems biology. While mass spectrometry has evolved over the last century into a highly effective analytical tool, there remain significant opportunities for innovation, allowing an even more diverse array of biological questions to be addressed. This proposal is centered on the development of new ionization methods for biological mass spectrometry to enable tissue imaging across several classes of biological molecules. The short term objective of this proposal is to further develop and fundamentally understand this innovative ionization method using real biological systems. These results will provide a solid foundation from which biological applications will directly benefit. In this mindset, we will develop and apply these new ionization methods to tissue imaging in model organisms to gain mechanistic insights into, 1) ischemic stroke; 2) wound healing; and 3) cardiometabolic disease. The long-term objective is to establish these new ionization methods as an enabling bioanalytical technology to effectively address questions in human health and disease. Public Description of Proposed Research Mass spectrometry (MS), the science related to the �������weighing of molecules�������, has had a profound impact on the study of human health and disease including cancer, heart disease, neurodegenerative diseases, neural development, and auto-immune diseases. A prerequisite of MS is to convert neutral molecules into charged species (ions) such that they can be �������weighed������� by the mass spectrometer and identified by advanced analytical techniques. The focus of this research is to develop new ionization methods allowing a more diverse array of contemporary biomedical questions to be addressed. This will include the imaging of tissues to ultimately provide new biological insights into stroke, wound healing and cardiometabolic disease.

Per- and polyfluoroalkyl substances (PFAS) are emerging as a major public health problem in North Carolina and across the United States. PFAS comprise a class of over 5,000 compounds. Their unique chemical properties have been harnessed to make consumer and industrial products more water, stain, and grease resistant; they are found in products as diverse as cosmetics and flame-retardants. PFAS are resistant to degradation, move easily through the environment, and accumulate in living organisms. Exposure to PFAS has been associated with health effects including cancer and toxicity to the liver, reproductive development, and thyroid and immune systems. Despite widespread detection in the environment and evidence of increasing human exposure, understanding about PFAS toxicity, its bioaccumulative potential in dietary sources such as aquatic organisms, and effective remediation remain notably understudied. The recent discovery by this proposed Center������������������s Deputy Director, Dr. Detlef Knappe, of widespread PFAS contamination in the Cape Fear River watershed in NC underscores that these compounds are in need of immediate investigation.. The goal of our Center is to advance understanding about the environmental and health impacts of PFAS. To meet this goal we are employing a highly trans-disciplinary approach that will integrate leaders in diverse fields (epidemiology, environmental science and engineering, biology, toxicology, immunology, data science, and advanced analytics); all levels of biological organization (biomolecule, pathway, cell, tissue, organ, model organism, human, and human population); state-of-the-art analytical technologies; cutting-edge data science approaches; a recognized track record in interdisciplinary, environmental health science (EHS) training; and well-established partnerships with government and community stakeholders.

Environmental toxins have a profound impact on human health, leading to in utero congenital defects, early childhood development and the incidence and progression of chronic adult disease. As well as being a major contributor to health burden overall, the heterogeneous distribution of environmental toxicants leads to health disparities between different groups. However, environmental toxicant exposures have a variable impact on individuals, and a substantial part of that variation is due to genetic effects. By studying these ����������������Gene x Environment��������������� (GxE) effects one can both quantify these effects and importantly gain mechanistic, molecular understanding of the biological pathways involved in toxicant response. We have assembled an international, multi-disciplinary team to exploit a unique resource, the wild-derived inbred Kiyosu panel of medaka (Japanese rice-paddy) fish, screening 10 environmental taxicats (chosen for their impact and chemical diversity) with 3 early development phenotypic endpoints (early heart beat deviation, heart developmental defect and cartilage development). The Kiyosu panel brings together the best features of both the Mouse Collaborative Cross and the Drosophila Reference Genome Panel (DRGP). Similar to the Mouse Collaborative Cross, the Kiyosu panel is in a model vertebrate with a broad range of genetics and genomics techniques. A critique of the Mouse Collaborative Cross is the relative lack of founding genetic diversity, and that the diversity is not representative of a wild population. Here the Kiyosu panel is similar to the DRGP being derived from a single diverse wild population and capturing the majority of its genetic diversity. The panel is already inbred and has full genome sequence. Preliminary data show strong GxE effects of selected chemicals in medaka, and careful statistical power analysis provides confidence that we can map individual loci using a directed F2 cross strategy from the lines showing extreme resistance/sensitivity. Having discovered individual GxE loci underlying differential toxicant response in this vertebrate, we will molecularly characterize the loci using in depth phenotyping, CRISPR genetic tools and RNAseq; we expect much of the genetic variation to be a variation in the regulation of xenobiotic pathways, so RNAseq will be particularly informative on the downstream impact of genetic variation. We will also integrate the information with both Mouse (Collaborative Cross) and human (UK BioBank) resources to translate this information to a human context.

The aims of this research are to evaluate the connection between VDD and obesity employing a bi-directional translational model in which clinical observations inform basic biological research in a zebrafish model, and mechanistic insights garnered from model organism research are applied to human research. Specifically, we will detemine if the obesognic effect of vitamin D deficiency (VDD) is a result of upregulation of endocannibinoid (EC) signaling as putativly discovered in zebrafish, and if this effect is operational in a cohort of children and adolescents exhibiting vitamin D deficiency in conjunction with hallmarks of obesity. If so this research will link two endocrine systems influnced by xenobitoics (endocannabinoid system and vitamin D signaling) and establish their role in the current epidemic of human obesity.

1. Specific Aims: Halogenated phenolic chemicals (HPCs, e.g. bromophenols, OH-PCBs, OH-BDEs) are common environmental contaminants of interest to the Superfund Research program with ubiquitous human exposure potential, and which are often classified as endocrine disruptors. In our previous funding period, we determined that 6-OH-BDE 47 was the most acutely toxic HPC investigated (LC50=130 nM) in early life stage exposures of zebrafish resulting in delayed development, loss of pigmentation, and skeletal deformities which was mediated at least in part by down regulation of thyroid hormone receptor beta (TR��������). TR receptors in conjunction with multiple nuclear receptors (PPARy, VDR, ER) facilitate a highly coordinated and orchestrated series of events governing the commitment and differentiation of mesenchymal stem cells to multiple mesenchymal lineages including osteoblasts (bone cells), chondrocytes (cartilage cells), adipocytes (fat cells) and others. Our laboratory also has recently demonstrated that select HPC������������������s adversely affect adipogenesis (development of adipocytes), at least partially mediated through agonism/antagonism of peroxisome proliferator activated receptor (PPAR). Furthermore, we have demonstrated that OH-BDEs, bromophenols and several high volume and ubiquitous aryl organophosphate esters (APEs) (e.g. triphenyl phosphate (TPP)) are agonists of PPAR��������. A theme that is emerging in this field is that toxicant exposures may alter gene regulatory networks that coordinate the balance of mesenchymal stem cell commitment towards adipogenic and osteochondral lineages. Here we expand upon our previous findings that exposure to select HPCs and APEs can lead to defined skeletal malformations and have the ability to modify adipogenic/osteochondral programs. We anticipate that the effects of HPCs and APEs on adipogenic and skeletal development may be occurring through TR and PPAR mediated pathways that converge on a similar phenotype. Here we test the hypothesis that early life exposure to HPCs and APEs (an particularly 6-OH-BDE-47, TBBPA and TPP) result in adverse effects on both osteochondral and adipogenic development through dysregulation of TR and PPAR signaling pathways. We propose to investigate this hypothesis using human cell cultures (in vitro) and the zebrafish model (in vivo). Our specific research aims are: SA1. Evaluate structure-activity relationships of HPCs and APEs using nuclear receptor structure-function assays including transactivation, ligand-receptor binding, ligand induced/repressed DNA binding and ligand induced/repressed NR-coregulator assays. More than 20 HPCs and APEs will be screened to identify the most potent compounds for further in vitro and in vivo testing proposed in the aims outlined below. At a minimum, we know that 6-OH-BDE-47, tetrabromobisphenol A (TBBPA) and TPP are potent nuclear receptor agonists and will be included for further assessments in the next two Specific Aims. SA2. Evaluate direct activational/repressive effects of 6-OH-BDE-47, TBBPA and TPP, individually and in environmentally relevant mixtures, on mesenchymal stem cell (MSC; human and/or mouse) proliferation and differentiation towards adipogenic and osteogenic lineages. MSC cultures will be dosed with TBBPA, TPP, 6-OH-BDE-47, and any other potent HPC/APE identified in SA1, to examine effects on proliferation and differentiation of adipocytes and osteocytes at the cellular level. SA3. Conduct in vivo (zebrafish) assessments using 6-OH-BDE-47, TBBPA and TPP, individually and in environmentally relevant mixtures, to quantify effects on impaired craniofacial and axial skeletal development and on adiposity. Transgenic fish models will be used including Twist:EGFP, Osx:mCherry, Col10a1:nlGFP in conjunction with histological and morphological staining for mineralized bone matrix and cartilage proteoglycans. In addition, effects on adiposity will be evaluated using histological and morphological staining for adipocyte/lipid accumulation.

In the proposed project, we will use the zebrafish model to investigate the behavioral consequences of environmental toxicants affecting retinoic acid receptors (RARs) during early neural development. Zebrafish provide an efficient model which bridges the gap between high throughput cell based in-vitro models which can quickly screen many compounds on a basic level and complex but slow and expensive investigations of humans in epidemiological studies and rodents in experimental studies. We will study RAR acting compounds because RAR signaling plays a critical role in proper formation of the neural tube/plate. Disruption of RAR signaling such as with the anticonvulsant drug valproic acid (VPA) has been shown to increase risk of Autism. We anticipate that even modest disruptions in RAR signaling will not result in dramatic alteration in neural organization until a later stage of neurodevelopment when the brainstem nuclei project rostrally to help organize later more rostral neurodevelopment. Then, the full phenotypic expression of the early neurotoxic injury may manifest in behavioral dysfunction. Here we propose that exposure to environmental toxicants affecting RAR signaling contribute to the increasing rates of Autism. We have found the zebrafish tests are sensitive to VPA and hypervitaminosis A induced impairments in social will be used to screen environmental compounds exhibiting RAR agonist activity which have been identified through mining recent Tox21/ToxCast high throughput screening data. Zebrafish offer a higher throughput and economic model system enabling analysis neurobehavioral consequences as affected by toxicants impacting RAR signaling during development thereby contributing to the risk of developmental neurobehavioral toxicity.

Environmental pollutants pose an incredible threat to animal and human health. Found ubiquitously in our daily lives and across the globe, individuals are exposed to these pollutants throughout their lives. They impact health at all stages of life, from in utero congenital defects to early childhood developmental defects and to the increased incidence and progression of chronic adult disease. Developmental toxicity is of particular interest in vertebrate models, since many of these pathways are contaminant targets and are highly conserved across vertebrate species (Leung et al. 2017). Most, if not all this work has come down to how contaminants interact with molecular pathways ������������������ what endogenous compound they mimic, what receptors they bind to, how they affect the amount of a certain product, etc. However, understanding these mechanisms is not enough to predict development in response to toxic exposures. Many of these contaminants have variable impact on individuals, which again is largely determined by genetic factors. This project proposes to answer some of the questions surrounding genetic effects on environmental exposures, better known as gene-environment interactions (G x E) using a unique model: a wild-derived inbred Kiyosu panel of medaka (Japanese rice-paddy) fish. Our partners have formed a panel consisting of over 100 inbred lines from this wild population and mapped out each of their genomes. As a multi-disciplinary, international collaboration we will be screening 10 environmental contaminants (chosen for impact and chemical diversity) with multiple early developmental, high dimensional, quantitative measurements on cardiac physiology, cardiac development and skeletal development, the latter of which I will be focusing.

1. Specific Aims: Halogenated phenolic chemicals (HPCs, e.g. bromophenols, OH-PCBs, OH-BDEs) are common environmental contaminants of interest to the Superfund Research program with ubiquitous human exposure potential, and which are often classified as endocrine disruptors. In our previous funding period, we determined that 6-OH-BDE 47 was the most acutely toxic HPC investigated (LC50=130 nM) in early life stage exposures of zebrafish resulting in delayed development, loss of pigmentation, and skeletal deformities which was mediated at least in part by down regulation of thyroid hormone receptor beta (TR��������). TR receptors in conjunction with multiple nuclear receptors (PPARy, VDR, ER) facilitate a highly coordinated and orchestrated series of events governing the commitment and differentiation of mesenchymal stem cells to multiple mesenchymal lineages including osteoblasts (bone cells), chondrocytes (cartilage cells), adipocytes (fat cells) and others. Our laboratory also has recently demonstrated that select HPC������������������s adversely affect adipogenesis (development of adipocytes), at least partially mediated through agonism/antagonism of peroxisome proliferator activated receptor (PPAR). Furthermore, we have demonstrated that OH-BDEs, bromophenols and several high volume and ubiquitous aryl organophosphate esters (APEs) (e.g. triphenyl phosphate (TPP)) are agonists of PPAR��������. A theme that is emerging in this field is that toxicant exposures may alter gene regulatory networks that coordinate the balance of mesenchymal stem cell commitment towards adipogenic and osteochondral lineages. Here we expand upon our previous findings that exposure to select HPCs and APEs can lead to defined skeletal malformations and have the ability to modify adipogenic/osteochondral programs. We anticipate that the effects of HPCs and APEs on adipogenic and skeletal development may be occurring through TR and PPAR mediated pathways that converge on a similar phenotype. Here we test the hypothesis that early life exposure to HPCs and APEs (an particularly 6-OH-BDE-47, TBBPA and TPP) result in adverse effects on both osteochondral and adipogenic development through dysregulation of TR and PPAR signaling pathways. We propose to investigate this hypothesis using human cell cultures (in vitro) and the zebrafish model (in vivo). Our specific research aims are: SA1. Evaluate structure-activity relationships of HPCs and APEs using nuclear receptor structure-function assays including transactivation, ligand-receptor binding, ligand induced/repressed DNA binding and ligand induced/repressed NR-coregulator assays. More than 20 HPCs and APEs will be screened to identify the most potent compounds for further in vitro and in vivo testing proposed in the aims outlined below. At a minimum, we know that 6-OH-BDE-47, tetrabromobisphenol A (TBBPA) and TPP are potent nuclear receptor agonists and will be included for further assessments in the next two Specific Aims. SA2. Evaluate direct activational/repressive effects of 6-OH-BDE-47, TBBPA and TPP, individually and in environmentally relevant mixtures, on mesenchymal stem cell (MSC; human and/or mouse) proliferation and differentiation towards adipogenic and osteogenic lineages. MSC cultures will be dosed with TBBPA, TPP, 6-OH-BDE-47, and any other potent HPC/APE identified in SA1, to examine effects on proliferation and differentiation of adipocytes and osteocytes at the cellular level. SA3. Conduct in vivo (zebrafish) assessments using 6-OH-BDE-47, TBBPA and TPP, individually and in environmentally relevant mixtures, to quantify effects on impaired craniofacial and axial skeletal development and on adiposity. Transgenic fish models will be used including Twist:EGFP, Osx:mCherry, Col10a1:nlGFP in conjunction with histological and morphological staining for mineralized bone matrix and cartilage proteoglycans. In addition, effects on adiposity will be evaluated using histological and morphological staining for adipocyte/lipid accumulation.

The research theme of Duke University������������������s Superfund Research Center (DUSRC) is early life exposure to hazardous chemicals and later life consequences. Over the last funding period, Project 2 investigated the effects of halogenated phenolic chemicals (HPCs, e.g. bromophenols, OH-PCBs, OH-BDEs) on pathways regulated by thyroid hormones and examined effects on development in later life stages using zebrafish as a model. We found that 6-OH-BDE 47 was the most acutely toxic HPC investigated (LC50=130 nM) in early life stage exposures to zebrafish, resulting in delayed development, loss of pigmentation, and skeletal deformities which was mediated at least in part by down regulation of thyroid hormone receptor beta (TR��������). Thyroid receptors in conjunction with multiple nuclear receptors (PPAR, VDR, ER) facilitate a highly coordinated and orchestrated series of events governing the commitment and differentiation of mesenchymal stem cells to multiple mesenchymal lineages including osteoblasts (bone cells), chondrocytes (cartilage cells), adipocytes (fat cells) and others. Our laboratory has recently demonstrated that select HPC������������������s and aryl organophosphate esters (AOPEs; e.g. Triphenyl phosphate) also adversely affect osteogenisis and adipogenesis (development of adipocytes), which are partially mediated through effects on the peroxisome proliferator activated receptor (PPAR). A theme that is emerging in this field is that early life toxicant exposures may alter gene regulatory networks that coordinate the balance of mesenchymal stem cell commitment towards adipogenic and osteochondral lineages. Here we expand upon our previous findings that exposure to select HPCs and AOPEs can lead to defined skeletal malformations and have the ability to modify adipogenic/osteochondral programs. We anticipate that the effects of HPCs and AOPEs on adipogenic and skeletal development may be occurring through TR and PPAR mediated pathways that converge on a similar phenotype. HPCs and AOPEs are hazardous substances, with high exposure potential indoors, and are of interest to the stakeholders of the Superfund Research program (e.g. EPA). Here we test the hypothesis that early life exposure to HPCs and AOPEs result in adverse effects on both osteochondral and adipogenic development through dysregulation of TR and PPAR signaling pathways. We propose to investigate this hypothesis using human cell cultures (in vitro) and the zebrafish model (in vivo). Our specific research aims are: 1. Evaluate direct activational/repressive effects of HPCs/AOPEs individually, and potentially with an environmentally relevant mixture, on mesenchymal stem cell (MSC; human and/or mouse) proliferation and differentiation towards adipogenic and osteogenic lineages. MSC cultures will be dosed with either TBBPA, 6-OH-BDE-47, Triphenyl phosphate (TPP), or an environmental mixture, to examine effects on proliferation and differentiation of adipocytes and osteocytes at the cellular level. 2. Conduct in vivo (zebrafish) assessments with the chemicals/mixtures used in Aim 1 to quantify effects on impaired craniofacial and axial skeletal development and on adiposity. Transgenic fish models will be used in conjunction with histological and morphological staining for mineralized bone matrix and cartilage proteoglycans. In addition, effects on adiposity will be evaluated using histological and morphological staining for adipocyte/lipid accumulation. 3. Collaborate with Projects 1 and 4 to determine if exposures conducted at levels below the threshold for effects on skeletal development result in effects on neurobehavior and bioenergetics. Zebrafish that were exposed in Aim 3 will be assessed for behavioral changes and effects on bioenergetics through collaborations with Projects 1 and 4. Mandates Targeted. This project directly addresses Superfund Mandates 1 and 2 (advanced techniques for assessment of the effect of hazardous substances on human health; methods to assess risks to human health). All aims will evaluate effects of hazardous substances on human health; w