Early life exposures can have important impacts on human development, and may influence our body composition. In early development, stem cells are directed by hormones to differentiate into various cell types. Thyroid and nuclear receptors facilitate the series of events, directing mesenchymal (embryonic) stem cells to differentiate into osteoblasts (bone cells), chondrocytes (cartilage cells), adipocytes (fat cells), and other cell types. This project focuses on the impacts of halogenated phenolic chemicals (HPCs) and aryl organophosphate esters (AOPEs) on pathways regulated by thyroid hormones.
AOPEs and HPCs are of interest because of their use as flame retardant compounds in many consumer products. Our research is exploring how exposure to HPCs and AOPEs during development can alter growth and skeletal development through dysregulation of specific thyroid and nuclear receptors: thyroid hormone receptor (TR) and the peroxisome proliferator activated receptor (PPAR).
Our previous research investigated the effects of HPCs, (e.g., bromophenols, hydroxylated brominated diphenyl ethers, or OH-BDEs) on pathways regulated by thyroid hormones and examined the effects on development in later life stages. We found that the compound 6-OH-BDE 47, a metabolite of a common flame retardant, was the most acutely toxic HPC investigated in terms of early life stage exposures to zebrafish. Exposure to 6-OH-BDE 47 resulted in delayed development, loss of pigmentation, and skeletal deformities in the fish, which was at least in part due to down-regulation of thyroid hormone receptor beta (TRβ). We recently completed that project and the final manuscript (Kollitz et al., 2018) has now been accepted for publication in Toxicological Sciences. We presented our results at the 2017 SETAC North America conference.
Thyroid hormone receptors, in conjunction with multiple nuclear receptors (including PPARy, VDR, and ER), facilitate a highly coordinated and orchestrated series of events that dictate the commitment and differentiation of mesenchymal stem cells. Again, these stem cells are the source of multiple cell lines, including osteoblasts, chondrocytes, adipocytes, and others. An emerging theme in our field of research is that early life toxicant exposures may alter gene regulatory networks that coordinate the balance of mesenchymal stem cell commitment towards adipogenic and osteochondral (bone/cartilage) cell lineages.
In our current project, we will expand upon our previous findings that exposure to select HPCs and AOPEs can lead to defined skeletal malformations through modification of highly regulated osteochondral and adipogenic transcriptional programs. Within this project, we are trying to understand how specific hazardous contaminants affect the developing organism, and specifically, how they affect the development of our bone and fat cells. Some of these contaminants have a chemical structure that is similar to hormones in our body, and they have the potential to bind and activate specific pathways in a way that can perturb development. In our research, we are using the zebrafish as a model to answer these questions.
Over the past year (2017-2018), we have demonstrated that these contaminants can bind proteins (nuclear receptors) that regulate thyroid hormones and bone development, and we found that the binding was very similar in both humans and zebrafish, demonstrating the utility of using zebrafish as a model. Of note, we found that some brominated flame retardant chemicals, which are used heavily today in consumer products, could strongly bind these proteins, which raise a number of questions about the ability of these contaminants to disrupt development.
We are evaluating our hypothesis using two models: human cell cultures (in vitro) and the zebrafish model (in vivo). Specifically, we use cell cultures to investigate the effects of these chemicals on proliferation and differentiation of mesenchymal stem cells, then we use the zebrafish as a whole organism model to quantify effects on craniofacial and skeletal development and adiposity (animal fat content). Results from this project will help us elucidate cross-talk and compensatory responses of chemicals that disrupt both TR and PPAR signaling pathways. Our study will also provide a hierarchical framework from the level of protein, to cell, to whole organism. Using this information, we can link mechanistic insights for HPC exposures with any acute developmental toxicities we observe in a small aquarium fish model of human disease (the zebrafish). Furthermore, we hope to identify key chemical structures that might impart greater bioactivity. We anticipate our approach will facilitate a broader conceptual understanding of the proposed adverse outcome pathways for HPCs and help inform human and environmental risk assessments.
Our primary research questions are listed below, followed by further descriptions of our research aims for this project:
- What are the impacts of HPCs/AOPEs individually, and as components of environmentally relevant mixtures, on differentiation of human and/or mouse mesenchymal stem cells toward fat cell- or bone cell-creating lineages?
- Using the same chemicals and mixtures from Aim 1, how do exposures affect normal bone and adipose tissue development in transgenic zebrafish models?
- In collaboration with Projects 1 and 4, examine how exposures to zebrafish (from Aim 2) below the threshold for skeletal development may affect neurobehavior or bioenergetics.
Aim 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 (fat cell-creating) and osteogenic (bone cell-creating) lineages. MSC cultures will be dosed with one of three flame retardant compounds (either TBBPA, 6-OH-BDE-47, or Triphenyl phosphate (TPP)), or an environmental mixture, to examine effects on proliferation and differentiation of adipocytes (fat cells) and osteocytes (bone cells) at the cellular level.
Aim 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 (tissue evaluation) and morphological (cell structure) staining for adipocyte/lipid accumulation.
Aim 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 2 will be assessed for behavioral changes and effects on bioenergetics through collaborations with Projects 1 and 4.
Project 2 Team:
Dr. Heather Stapleton, PI (Stapleton Lab)
Dr. Seth Kullman, Co-PI (Kullman Lab)
Dr. Lee Ferguson, Co-PI (Ferguson Lab)
Erin Kollitz, PhD, Postdoctoral Research Associate
Alli Phillips, PhD student
Matt Ruis, PhD student
Stacy Shkoda, PhD student (NCSU)