Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is an organophosphate flame retardant used around the world. Within zebrafish, we previously showed that initiation of TDCIPP exposure during cleavage (0.75 h post-fertilization, hpf) results in epiboly disruption at 6 hpf, leading to dorsalized embryos by 24 hpf, a phenotype that mimics the effects of dorsomorphin (DMP), a bone morphogenetic protein (BMP) antagonist that dorsalizes embryos in the absence of epiboly defects. The objective of this study was to (1) investigate the role of BMP signaling in TDCIPP-induced toxicity during early embryogenesis, (2) identify other pathways and processes targeted by TDCIPP, and (3) characterize the downstream impacts of early developmental defects. Using zebrafish as a model, we first identified a sensitive window for TDCIPP-induced effects following exposure initiation at 0.75 hpf. We then investigated the effects of TDCIPP on the transcriptome during the first 24 h of development using mRNA sequencing and amplicon sequencing. Finally, we relied on whole-mount immunohistochemistry, dye-based labeling, and morphological assessments to study abnormalities later in embryonic development. Overall, our data suggest that the initiation of TDCIPP exposure during early blastula alters the normal trajectory of early embryogenesis by inducing gastrulation defects and aberrant germ-layer formation, leading to abnormal tissue and organ development within the embryo.

Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) is a high-production-volume organophosphate flame retardant (OPFR) that induces epiboly defects during zebrafish embryogenesis, leading to the disruption of dorsoventral patterning. Therefore, the objectives of this study were to (1) identify the potential mechanisms involved in TDCIPP-induced epiboly defects and (2) determine whether coexposure to triphenyl phosphate (TPHP)-an OPFR commonly detected with TDCIPP-enhances or mitigates epiboly defects. Although TDCIPP-induced epiboly defects were not associated with adverse impacts on cytoskeletal protein abundance in situ, the coexposure of embryos to TPHP partially blocked TDCIPP-induced epiboly defects. As nuclear receptors are targets for both TPHP and TDCIPP, we exposed the embryos to TDCIPP in the presence or absence of 69 nuclear receptor ligands and, similar to TPHP, found that ciglitazone (a peroxisome proliferator-activated receptor γ agonist) and 17β-estradiol (E2; an estrogen receptor α agonist) nearly abolished TDCIPP-induced epiboly defects. Moreover, E2 and ciglitazone mitigated TDCIPP-induced effects on CpG hypomethylation within the target loci prior to epiboly, and ciglitazone altered TDCIPP-induced effects on the abundance of two polar metabolites (acetylcarnitine and cytidine-5-diphosphocholine) during epiboly. Overall, our results point to a complex interplay among nuclear receptor ligands, cytosine methylation, and the metabolome in both the induction and mitigation of epiboly defects induced by TDCIPP.

Niclosamide is an antihelminthic drug used worldwide for the treatment of tapeworm infections. Recent drug repurposing screens have highlighted the broad bioactivity of niclosamide across diverse mechanisms of action. As a result, niclosamide is being evaluated for a range of alternative drug-repurposing applications, including the treatment of cancer, bacterial infections, and Zika virus. As new applications of niclosamide will require non-oral delivery routes that may lead to exposure in utero, it is important to understand the mechanism of niclosamide toxicity during early stages of embryonic development. Previously, we showed that niclosamide induces a concentration-dependent delay in epiboly progression in the absence of effects on oxidative phosphorylation - a well-established target for niclosamide. Therefore, the overall objective of this study was to further examine the mechanism of niclosamide-induced epiboly delay during zebrafish embryogenesis. Based on this study, we found that (1) niclosamide exposure during early zebrafish embryogenesis resulted in a decrease in yolk sac integrity with a concomitant decrease in the presence of yolk sac actin networks and increase in cell size; (2) within whole embryos, niclosamide exposure did not alter non-polar metabolites and lipids, but significantly altered amino acids specific to aminoacyl-tRNA biosynthesis; (3) niclosamide significantly altered transcripts related to translation, transcription, and mRNA processing pathways; and (4) niclosamide did not significantly alter levels of rRNA and tRNA. Overall, our findings suggest that niclosamide may be causing a systemic delay in embryonic development by disrupting the translation of maternally-supplied mRNAs, an effect that may be mediated through disruption of aminoacyl-tRNA biosynthesis.