High-resolution mass spectrometry reveals environmentally relevant uptake, elimination, and 1 metabolic alterations following early embryonic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin 2 in zebrafish 3

Dioxin and dioxin-like compounds are ubiquitous environmental contaminants that induce 23 toxicity by binding to the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. The zebrafish model has been used to define the developmental toxicity observed following 25 exposure to exogenous AHR ligands such as the potent agonist 2,3,7,8-tetrachlorodibenzo-p- 26 dioxin (dioxin, TCDD). While the model has successfully identified cellular targets of TCDD and molecular mechanisms mediating TCDD-induced phenotypes, fundamental information such as 28 the body burden produced by standard exposure paradigms is still unknown. We performed 29 targeted gas chromatography (GC) high-resolution mass spectrometry (HRMS) in tandem with 30 non-targeted liquid chromatography (LC) HRMS to quantify TCDD uptake, model the elimination 31 dynamics of TCDD, and determine how TCDD exposure affects the zebrafish metabolome. We 32 found that 10 ppb, 1 ppb, and 50 ppt waterborne exposures during early embryogenesis 33 produced environmentally relevant body burden of TCDD: 38 ± 4.34, 26.6 ± 1.2, and 8.53 ± 34 0.341 pg/embryo, respectively, at 24 hours post fertilization. In addition, we discovered that 35 TCDD exposure was associated with the dysregulation of several metabolic pathways that are 36 critical for brain development and function including glutamate metabolism, chondroitin sulfate 37 biosynthesis, and tyrosine metabolism pathways. Together, these data demonstrate that 38 existing exposure paradigms produce environmentally relevant body burdens of TCDD in 39 zebrafish and provide insight into the biochemical pathways impacted by toxicant-induced AHR 40 activation. 41 of exposure in critical data chromatography (GC) (HRMS) quantify TCDD zebrafish chromatography (LC) HRMS metabolomics how metabolome. one-hour TCDD exposures at 4 hours post (hpf) three that are routinely used in zebrafish TCDD exposure studies: 50 parts-per-trillion (ppt), and 10 The 50 ppt exposure is used to model sublethal has been applied in transgenerational TCDD exposure studies, phenotypes metabolomic analysis quantify TCDD concentrations in the zebrafish embryo at key developmental timepoints. We found that TCDD is eliminated by exponential decay after a 1 ppb and 10 ppb 508 exposure, while after a lower, 50 ppt dose, a linear model better captures TCDD elimination. Embryonic and larval body burdens of TCDD for both doses were previously unknown. Therefore, this study provides essential information that helps contextualize studies using this common dosing paradigm. We used non-targeted high-resolution LC-HRMS to detect metabolites in the zebrafish embryo and then performed metabolomic pathway analysis to identify known metabolic pathways that were affected by TCDD exposure. Zebrafish continue to be a powerful model for studying the health effects of dioxin exposure as metabolomics revealed pathways that are not detectable in serum, blood or urine samples, such as bile acid biosynthesis. Further, neurotransmitter pathways were associated with TCDD exposure, demonstrating that the study of the zebrafish metabolome after TCDD exposure may reveal previously overlooked effects of dioxin exposure on the health of humans and aquatic species environment.