Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that heterodimerizes with retinoid X receptor (RXR), binds to PPAR response elements (PPREs), and activates transcription of genes involved in adipogenesis, fatty acid storage, and glucose metabolism. PPARγ-specific pharmaceuticals (e.g., thiazolidinediones) have been developed to treat Type II diabetes within human populations. However, in vitro and in vivo (rodent) studies have demonstrated that PPARγ can also be activated by environmental chemicals. Due to the ubiquitous presence of these environmental chemicals, they pose a potential concern for those chronically exposed via inhalation or ingestion of contaminated dust. However, while the potential role of PPARγ in regulating later stages of development has been explored, it is unclear whether 1) exposure to PPARγ agonists may alter early embryonic developmental processes and 2) downstream impacts on cell signaling and physiology are directly associated with PPARγ activation by drugs and environmental chemicals. Therefore, the primary objectives of this dissertation are to 1) test the hypothesis that exposure to a human PPARγ agonist (ciglitazone) disrupts dorsoventral patterning during early embryonic development within zebrafish, 2) map genomic, transcriptomic, and lipidomic changes in human hepatocarcinoma (HepG2) cells after exposure to a reference PPARγ agonist (ciglitazone) and antagonist (GW 9662), and 3) test the hypothesis that exposure to environmental chemicals (tetrabromobisphenol A and tetrachlorobisphenol A) – suspected PPARγ agonists – results in PPARγ-mediated transcriptional and lipidomic changes within HepG2 cells. For Aim 1, using ciglitazone to activate PPARγ and a pparγ-specific morpholino to knockdown pparγ, we found that ciglitazone affects dorsoventral patterning in developing zebrafish embryos in a PPARγ-independent manner. For Aim 2, using chromatin immunoprecipitation-sequencing, mRNA-sequencing, neutral lipid staining, and lipidomics, we mapped genomic, transcriptomic, and lipidomics changes following exposure to a ciglitazone or GW 9662, but did not find changes at one level to be predictive of changes at other levels. For Aim 3, TBBPA and TCBPA exposure did not result in downstream PPARγ-dependent transcriptomic or neutral lipid changes. Overall, our findings further our understanding about how exogenous PPARγ ligands may alter early embryonic development and affect lipid homeostasis within human cells.

Organophosphate esters (OPEs) are a class of semi-volatile organic compounds that are used as flame retardants, plasticizers, and anti-foaming agents. Due to their ubiquitous use and ability to migrate out of end-use products, elevated concentrations of OPEs have been detected within indoor dust samples, a prevalent source for human exposure. Similar to the built environment, organophosphate esters are introduced into vehicles as plasticizers and flame retardants and have the potential to migrate and accumulate within vehicle dust. While human exposure to OPEs via indoor dust is well characterized, more information is needed to address the extent of OPE exposure and subsequent risk with time spent in vehicles and understand mechanisms underlying OPE-specific toxicity during early development. Therefore, the primary objectives of this dissertation are to 1) increase our understanding of the mechanisms of developmental toxicity for triphenyl phosphate (TPHP), a high-production volume OPE, 2) measure human exposure to OPEs within a population that spends a significant amount of time in personal transportation, and 3) calculate the risk associated with ingestion of tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), another high-production volume OPE, within car interiors. For Aim 1, using whole-embryo exposures and a combination of mRNA-sequencing, phenotypic assessments, neutral lipid staining, and metabolomics, TPHP was shown to result in adverse effects on the liver, lipid abundance, and osmoregulation within the developing zebrafish. For Aim 2, using silicone wristbands to monitor personal OPE exposure within a subset of commuter vs. non-commuter undergraduate students at the University of California, Riverside (UCR), we identified that longer commutes were associated with increased TDCIPP exposure. Building on the results of Aim 2, in Aim 3 we demonstrated that a reduction in car dashboard dust does not affect increased TDCIPP exposure as a result of longer time spent in vehicles. Lastly, for Aim 4, a risk assessment identified that the ingestion of TDCIPP from car interior dust does not pose a cancer risk for commuters in California. Overall, our findings contribute to our understanding of human OPE exposure in relation to commute times and how TPHP – a ubiquitous OPE – may alter embryonic development.

Triphenyl phosphate (TPHP) is an unsubstituted aryl phosphate ester used as a flame retardant and plasticizer within the United States. Using zebrafish as a model, the objectives of this study were to rely on (1) mRNA-sequencing to uncover pathways disrupted following embryonic TPHP exposure and (2) high-content screening to identify nuclear receptor ligands that enhance or mitigate TPHP-induced cardiotoxicity. Based on mRNA-sequencing, TPHP exposure from 24 to 72-h postfertilization (hpf) resulted in a concentration-dependent increase in the number of transcripts significantly affected at 72 hpf, and pathway analysis revealed that 5 out of 9 nuclear receptor pathways were associated with the retinoid X receptor (RXR). Based on a screen of 74 unique nuclear receptor ligands as well as follow-up experiments, 2 compounds—ciglitazone (a peroxisome proliferator-activated receptor gamma, or PPARγ, agonist) and fenretinide (a pan-retinoic acid receptor, or RAR, agonist)—reliably mitigated TPHP-induced cardiotoxicity in the absence of effects on TPHP uptake or metabolism. As these data suggested that TPHP may be activating RXR (a heterodimer for both RARs and PPARγ), we co-exposed embryos to HX 531—a pan-RXR antagonist—from 24 to 72 hpf and, contrary to our hypothesis, found that co-exposure to HX 531 significantly enhanced TPHP-induced cardiotoxicity. Using a luciferase reporter assay, we also found that TPHP did not activate nor inhibit chimeric human RXRα, RXRβ, or RXRγ, suggesting that TPHP does not directly bind nor interact with RXRs. Overall, our data suggest that TPHP may interfere with RXR-dependent pathways involved in cardiac development

For many compounds currently used in commerce, there are minimal developmental toxicity data available. Additionally, current developmental toxicity testing guidelines require the use of rodents, making them expensive, time consuming, and inefficient to address existing data gaps. Therefore, alternatives to conventional animal testing – such as cell-free assays, cell-based assays, and non-protected stages of non-mammalian models – are needed to support the screening and prioritization of chemicals for developmental toxicity testing. Zebrafish offer one of the most promising alternative and cost-effective vertebrate models to support drug discovery and toxicity testing. Their small size, rapid development, and conservation of early developmental processes with mammals (including humans), make the zebrafish embryo an ideal model for the identification of compounds that disrupt the normal trajectory of early embryonic development. Therefore, the overall goal of this research is to leverage early, non-protected life stages of zebrafish embryos to identify compounds that may be adversely impacting early development. For Aim 1, using high-content screening techniques, embryonic behavior was examined as a potential readout for detection of compounds adversely impacting the development and function of the nervous system. For Aim 2, the effects of niclosamide – a widely used anthelmintic that was identified from our high-content screen – on early embryonic development was assessed using a multipronged approach at multiple levels of biological organization. For Aim 3, based on our findings within Specific Aim 2, the mechanism of niclosamide-induced developmental toxicity was further examined using a combination of whole-embryo assessments and human embryonic stem cell-based assays. Overall, our findings highlight the utility of embryonic zebrafish as a physiologically-intact, non-mammalian model for 1) rapid chemical screening and prioritization for developmental toxicity testing; 2) discovering biologically active yet understudied chemicals; and 3) investigating mechanisms of developmental toxicity.

Organophosphate flame retardants (OPFR) are semi-volatile additive flame retardants that are found in a variety of products that contain polyurethane foam, textiles, and plasticizers. As products age, these chemicals can leach from their end-use products into the air and adhere to certain environmental media such as dust. Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is an organophosphate ester-based flame retardant widely used within the United States. TDCIPP has been detected in breast milk, hair follicles, and placental tissue in biomonitoring studies, and previous research has found a significant association between TDCIPP exposure and adverse health effects within human populations. Therefore, the global presence of TDCIPP within indoor air and dust poses a potential public health concern, particularly for pregnant mothers who may be exposed to TDCIPP-contaminated dust or air via ingestion or inhalation. However, the potential mechanisms underlying TDCIPP-induced effects on DNA methylation, epigenome plasticity, and cellular metabolism during early embryonic development has yet to be fully understood. Moreover, little is known about whether 1) TDCIPP alters key developmental timepoints regulated by DNA methylation and 2) TDCIPP-induced impacts on DNA methylation impact viability and metabolism within human embryonic cells. Within Chapter 2, we utilized immunohistochemistry, high-content screening, and in situ-based protocols to develop a method that allows us to track 5-mC across multiple stages of development and dose concentrations within early embryogenesis of zebrafish embryos. Within Chapter 3, we utilized bisulfite amplicon sequencing (BSAS), bioinformatics, in vitro assays, and in situ-based protocols to understand how TDCIPP exposure impacts cytosine methylation and 5-mC formation within developing zebrafish embryos. Within Chapter 4, we relied on human embryonic kidney (HEK293) cells to determine whether TDCIPP affects cell viability, reactive oxygen species (ROS) production, global 5-mC methylation, cell membrane integrity, mitochondria abundance, and intracellular ATP production by utilizing a combination of in vitro, in situ, and real-time cell analysis methods. Overall, the findings from this dissertation have increased our understanding about how TDCIPP alters early embryonic development, cellular metabolism, and global DNA/RNA methylation within zebrafish and human cells.