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Pericardial edema - fluid accumulation within the pericardium - is a frequently observed malformation in zebrafish embryo-based chemical toxicity screens. We recently discovered that the severity of triphenyl phosphate (TPHP)-induced pericardial edema was dependent on the ionic strength of exposure media. TPHP is an aryl phosphate ester (APE) widely used as a plasticizer and flame retardant. APEs are characterized by having one or more aryl groups bound to a phosphate center, with TPHP containing only unsubstituted aryl groups. Therefore, the objective of this study was to begin investigating whether, similar to TPHP, pericardial edema induced by other structurally related APEs is dependent on the ionic composition of exposure media. We first mined the peer-reviewed literature to identify other APEs that 1) induced pericardial edema in zebrafish embryos within a minimum of three peer-reviewed publications, and 2) demonstrated a statistically significant induction of pericardial edema in at least 70 % of the studies evaluated. Based on this meta-analysis, we identified four other APEs that caused pericardial edema in zebrafish embryos: isopropylated triphenyl phosphate (IPTPP), cresyl diphenyl phosphate (CDP), tricresyl phosphate (TMPP), and 2-ethylhexyl diphenyl phosphate (EDHPHP). Using TPHP as a positive control and pericardial edema as a readout, we developed concentration-response curves for all four APEs based on static exposure from 24 to 72 h post-fertilization (hpf). We then conducted co-exposures with D-Mannitol (an osmotic diuretic) and exposures within reverse osmosis (RO) water determine whether the ionic composition of exposure media mitigated APE-induced pericardial edema at 72 hpf. Using pericardial edema as an endpoint, the approximate EC 50 s for TPHP (positive control), IPTPP, CDP, TMPP, and EDHPHP were 6.25, 3.125, 3.125, 25, and 100 µM, respectively, based on exposure from 24 to 72 hpf. Interestingly, similar to our findings with TPHP, co-exposure with D-Mannitol and exposure within ion-deficient water significantly mitigated IPTPP- CDP-, TMPP-, and EDHPHP-induced pericardial edema in zebrafish embryos, suggesting that chemically-induced pericardial edema may be 1) dependent on the ionic composition of exposure media and 2) driven by a disruption in osmoregulation across the embryonic epidermis. Therefore, similar to other assay parameters, our findings underscore the need to standardize the osmolarity of exposure media in order to minimize the potential for false positive/negative hits in zebrafish embryo-based chemical toxicity screens conducted around the world., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In the United States and abroad, ortho-phthalates and non-ortho-phthalate plasticizers continue to be used within a diverse array of consumer products. Prior California-specific biomonitoring programs for ortho-phthalates have focused on rural, agricultural communities and, to our knowledge, these programs have not measured the potential for exposure to non-ortho-phthalate plasticizers. Therefore, the potential for human exposure to ortho-phthalates and non-ortho-phthalate plasticizers have not been adequately addressed in regions of California that have higher population density. Since there are numerous sources of ortho-phthalates and non-ortho-phthalate plasticizers in population-dense, urban regions, the objective of this study was to leverage silicone wristbands to quantify aggregate ortho-phthalate and non-ortho-phthalate plasticizer exposure over a 5-day period across two different cohorts (2019 and 2020) of undergraduate students at the University of California, Riverside (UCR) that commute from all over Southern California. Based on 5 d of aggregate exposure across two different cohorts, total ortho-phthalate plus non-ortho-phthalate plasticizer concentrations ranged, on average, from ∼100,000-1,000,000 ng/g. Based on the distribution of individual ortho-phthalate and non-ortho-phthalate plasticizer concentrations, the concentrations of di-isononyl phthalate (DiNP, a high molecular weight ortho-phthalate), di (2-ethylhexyl) phthalate (DEHP, a high molecular weight ortho-phthalate), and di-2-ethylhexyl terephthalate (DEHT, a non-ortho-phthalate plasticizer) detected within wristbands were higher than the remaining seven ortho-phthalates and non-ortho-phthalate plasticizers measured, accounting for approximately 94-97% of the total mass depending on the cohort. Overall, our findings raise concerns about chronic DiNP, DEHP, and DEHT exposure in urban, population-dense regions throughout California., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a widely used, additive flame retardant that migrates from end-use products, leading to ubiquitous exposure of humans around the world. However, little is known about whether TDCIPP disrupts the physiology of human embryonic cells. Therefore, the objective of this study was to determine whether TDCIPP alters cell viability, cellular metabolism, cytosine methylation, and reactive oxygen species (ROS) levels within human embryonic kidney (HEK293) cells. Relative to vehicle controls, TDCIPP (0.015-0.1225 µM) resulted in a concentration-dependent increase in cell viability, a finding that was driven by an increase in relative ATP abundance. Interestingly, TDCIPP (0.061-0.98 µM) increased the rate of glycolysis - an adaptive mechanism consistent with the Warburg effect exhibited by tumorigenic cells. Moreover, relative to vehicle-treated cells, TDCIPP (0.245-15.63 µM) exposure for 48 h (but not 24 h) resulted in a significant, concentration-dependent decrease in ROS in situ, and TDCIPP (0.245 µM) exposure significantly increased carnosine within the histidine metabolism pathway. However, TDCIPP did not affect global 5-methylcytosine (5-mC) methylation (0.015-15.63 µM), cell membrane integrity (0.061-0.98 µM), nor the abundance of mitochondria (0.061-1.95 µM). Overall, our findings with TDCIPP point to a novel mechanism of action that may be relevant to human embryonic stem cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Triphenyl phosphate (TPHP) - a widely used organophosphate-based flame retardant - blocks cardiac looping during zebrafish development in a concentration-dependent manner, a phenotype that is dependent on disruption of embryonic osmoregulation and pericardial edema formation. However, it's currently unclear whether (1) TPHP-induced effects on osmoregulation are driven by direct TPHP-induced injury to the embryonic epidermis and (2) whether TPHP-induced pericardial edema is reversible or irreversible following cessation of exposure. Therefore, the objectives of this study were to determine whether TPHP-induced pericardial edema is reversible and whether TPHP causes injury to the embryonic epidermis by quantifying the number of DAPI-positive epidermal cells and analyzing the morphology of the yolk sac epithelium using scanning electron microscopy. First, we found that exposure to 5 μM TPHP from 24-72 h post-fertilization (hpf) did not increase prolactin - a hormone that regulates ions and water levels - in embryonic zebrafish, whereas high ionic strength exposure media was associated with elevated levels of prolactin. Second, we found that exposure to 5 μM TPHP from 24-72 hpf did not decrease DAPI-positive epidermal cells within the embryonic epithelium, and that co-exposure with 2.14 μM fenretinide - a synthetic retinoid that promotes epithelial wound repair - from 24-72 hpf did not mitigate the prevalence of TPHP-induced epidermal folds within the yolk sac epithelium when embryos were exposed within high ionic strength exposure media. Finally, we found that the pericardial area and body length of embryos exposed to 5 μM TPHP from 24-72 hpf were similar to vehicle-treated embryos at 120 hpf following transfer to clean water and depuration of TPHP from 72-120 hpf. Overall, our findings suggest that (1) the ionic strength of exposure media may influence the baseline physiology of zebrafish embryos; (2) TPHP does not cause direct injury to the embryonic epidermis; and (3) TPHP-induced effects on pericardial area and body length are reversible 48 h after transferring embryos to clean water., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Pericardial edema is commonly observed in zebrafish embryo-based chemical toxicity screens, and a mechanism underlying edema may be disruption of embryonic osmoregulation. Therefore, the objective of this study was to identify whether triphenyl phosphate (TPHP) - a widely used aryl phosphate ester-based flame retardant - induces pericardial edema via impacts on osmoregulation within embryonic zebrafish. In addition to an increase in TPHP-induced microridges in the embryonic yolk sac epithelium, an increase in ionic strength of exposure media exacerbated TPHP-induced pericardial edema when embryos were exposed from 24 to 72 h post-fertilization (hpf). However, there was no difference in embryonic sodium concentrations in situ within TPHP-exposed embryos relative to embryos exposed to vehicle (0.1% DMSO) from 24 to 72 hpf. Interestingly, increasing the osmolarity of exposure media with mannitol (an osmotic diuretic which mitigates TPHP-induced pericardial edema) and increasing the ionic strength of the exposure media (which exacerbates TPHP-induced pericardial edema) did not affect embryonic doses of TPHP, suggesting that TPHP uptake was not altered under these varying experimental conditions. Overall, our findings suggest that TPHP-induced pericardial edema within zebrafish embryos is dependent on the ionic strength of exposure media, underscoring the importance of further standardization of exposure media and embryo rearing protocols in zebrafish-based chemical toxicity screening assays., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Exposure to cigarette smoke represents the largest source of preventable death and disease in the United States. This may be in part due to the nature of the delayed harmful effects as well as the lack of awareness of the scope of harm presented by these products. The presence of "light" versions further clouds the harmful effects of tobacco products. While active smoking in expectant mothers may be reduced by educational and outreach campaigns, exposure to secondhand smoke is often involuntary yet may harm the developing embryo. In this study, we show that the main component of secondhand smoke, sidestream cigarette smoke, from several brands, including harm-reduction versions, triggered unsuccessful hatching at 3 dpf and reduced overall survival at 6 dpf in developing zebrafish. At non-lethal concentrations, craniofacial defects with different severity based on the cigarette smoke extract were noted by 6 dpf. All tested products, including harm-reduction products, significantly impacted cartilage formation and/or bone mineralization in zebrafish embryos, independent of whether the bones/cartilage formed from the mesoderm or neural crest. Together, these results in a model system often used to detect embryonic malformations imply that exposure of a woman to secondhand smoke while pregnant may lead to mineralization issues in the skeleton of her newborn, ultimately adding a direct in utero association to the increased fracture risk observed in children of mothers exposed to cigarette smoke.

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) is an organophosphate ester-based flame retardant widely used within the United States. Within zebrafish, initiation of TDCIPP exposure at 0.75 h post-fertilization (hpf) reliably disrupts cytosine methylation from cleavage (2 hpf) through early-gastrulation (6 hpf). Therefore, the objective of this study was to determine whether TDCIPP-induced effects on cytosine methylation persist beyond 6 hpf. First, we exposed embryos to vehicle or TDCIPP from 0.75 hpf to 6, 24, or 48 hpf, and then conducted bisulfite amplicon sequencing of a target locus (lmo7b) using genomic DNA derived from whole embryos. Within both vehicle- and TDCIPP-treated embryos, CpG methylation was similar at 6 hpf and CHG/CHH methylation were similar at 24 and 48 hpf (relative to 6 hpf). However, relative to 6 hpf within the same treatment, CpG methylation was lower within vehicle-treated embryos at 48 hpf and TDCIPP-treated embryos at 24 and 48 hpf - an effect that was driven by acceleration of CpG hypomethylation. Similar to our previous findings with DNA methyltransferase, we found that, even at high μM concentrations, TDCIPP had no effect on zebrafish and human thymine DNA glycosylase activity (a key enzyme that decreases CpG methylation), suggesting that TDCIPP-induced effects on CpG methylation are not driven by direct interaction with thymine DNA glycosylase. Finally, using 5-methylcytosine (5-mC)-specific whole-mount immunochemistry and automated imaging, we found that exposure to TDCIPP increased 5-mC abundance within the yolk of blastula-stage embryos, suggesting that TDCIPP may impact cytosine methylation of maternally loaded mRNAs during the maternal-to-zygotic transition. Overall, our findings suggest that TDCIPP disrupts the trajectory of cytosine methylation during zebrafish embryogenesis, effects which do not appear to be driven by direct interaction of TDCIPP with key enzymes that regulate cytosine methylation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) - both halogenated bisphenol (BPA) analogues - are suspected ligands of peroxisome proliferator-activated receptor gamma (PPARγ). While previous studies have shown that TBBPA and TCBPA activate PPARγ within cell-free assays, the downstream effects of TBBPA- and TCBPA-induced PPARγ activation on cellular transcription and physiology have not been thoroughly investigated. Therefore, the objective of this study was to determine whether exposure to TBBPA or TCBPA (either alone or in combination) alters levels of neutral lipids and fatty acid synthase (FASN) - an enzyme that catalyzes synthesis of long-chain saturated fatty acids - within intact cells in a PPARγ-dependent manner. For this study, we relied on human hepatocellular carcinoma (HepG2) cells as a model since these liver cells express basal levels of PPARγ and have been used to study lipoprotein metabolism and regulation of drug metabolizing enzymes. Although exposure to TBBPA and TCBPA alone did not affect cell viability nor neutral lipid and FASN levels in a concentration-dependent manner, exposure to binary mixtures of TBBPA and TCBPA resulted in a concentration-dependent decrease in cell viability in the absence of concentration-dependent effects on neutral lipid and FASN levels. Interestingly, exposure to TBBPA or TCBPA alone or as a mixture enhanced the effects of a reference PPARγ agonist (ciglitazone) and antagonist (GW 9662) on cell viability (but not neutral lipid levels), suggesting that these two halogenated BPA analogues may interact synergistically with ciglitazone and GW 9662 to induce cytotoxicity. However, overexpression of PPARγ did not mitigate nor enhance the effects of TBBPA - a potent PPARγ ligand predicted by ToxCast's cell-free competitive binding assays - on cell viability, neutral lipid levels, nor the cellular transcriptome. Overall, our findings suggest that halogenated BPA analogues such as TCBPA and TBBPA induce toxicity within HepG2 cells in a PPARγ-independent manner., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Organophosphate esters (OPEs) have been detected within car interior dust, suggesting that the indoor microenvironment of vehicles may represent a potential route of human exposure to OPEs. We recently showed that people with longer commutes are exposed to higher concentrations of tris(1,3-dichloro-2-isopropyl)phosphate (TDCIPP) - a widely used OPE - and other studies have suggested that dust removal may lead to lower exposure to chemicals. Therefore, the overall objective of this study was to determine if a decrease in interior car dust results in mitigation of personal OPE exposure. Participants (N = 49) were asked to wear silicone wristbands, and a subset of them wiped interior parts at the front of their vehicles prior to one study week (N = 25) or both study weeks (N = 11). There were no significant differences in total OPE concentrations (77.79-13,660 ng/g) nor individual OPE concentrations (0.04-4852.81 ng/g) across the different wiping groups nor in relation to participant residence ZIP codes and AC/Heater usage. These findings suggest that higher exposure to TDCIPP for participants with longer commutes may be independent of dust located on interior parts at the front of the vehicle. Therefore, our study demonstrates that there is a need for research on the potential contribution of other sources of TDCIPP exposure within car interiors., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Dysregulation of microRNA (miRNA, miR) by environmental stressors influences the transcription of mRNA which may impair organism development and/or lead to adverse physiological outcomes. Early studies evaluating the effects of oil on developmental toxicity in early life stages of fish showed that reductions in expression of miR-203a were associated with enhanced expression of downstream mRNAs that predicted altered eye development, cardiovascular disease, and improper fin development. To better understand the effects of miR-203a inhibition as an outcome of oil-induced toxicity in early life stage (ELS) fish, embryonic zebrafish were injected with an miR-203a inhibitor or treated with 3.5 µM phenanthrene (Phe) as a positive control for morphological alterations of cardiovascular and eye development caused by oil. Embryos treated with Phe had diminished levels of miR-203a at 7 and 72 h after injection. Embryos treated with the miR-203a inhibitor and Phe exhibited a reduced heart rate by 48 h post fertilization (hpf), with an increased incidence of developmental deformities (including pericardial edema, altered eye development, and spinal deformities) and reduced caudal fin length by 72 hpf. There were significant reductions in lens and eye diameters in 120 hpf miR-203a-inhibitor and Phe-treated fish, as well as a significantly reduced number of eye saccades, determined by an optokinetic response (OKR) behavioral assay. The expression of vegfa , which is an important activator during neovascularization, was significantly upregulated in embryos receiving miR-203a inhibitor injections by 7 and 72 hpf with increased trends in vegfa expression in 72 hpf larvae treated with Phe. There were decreasing trends in crx , neurod1 , and pde6h expression by 72 hpf in miR-203a inhibitor and Phe treatments, which are involved in photoreceptor function in developing eyes and regulated by miR-203a. These results suggest that an inhibition of miR-203a in ELS fish exhibits an oil-induced toxic response that is consistent with Phe treatment and specifically impacts retinal, cardiac, and fin development in ELS fish., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Triphenyl phosphate (TPHP) is an organophosphate ester-based plasticizer and flame retardant. The objective of this study was to identify the potential role of epidermal ionocytes in mediating TPHP-induced pericardial edema within zebrafish embryos. Exposure to TPHP from 24 to 72 h post fertilization (hpf) resulted in a significant increase in pericardial edema and the number of ionocytes at 72 hpf relative to time-matched embryos treated with vehicle. In addition, co-exposure of embryos to mannitol (an osmotic diuretic) blocked TPHP-induced pericardial edema and effects on ionocyte abundance. However, knockdown of ATPase1a1.4 - an abundant Na + /K + -ATPase localized to epidermal ionocytes - mitigated TPHP-induced effects on ionocyte abundance but not pericardial edema, whereas co-exposure of embryos to ouabain - a Na + /K + -ATPase inhibitor - enhanced TPHP-induced pericardial edema but not ionocyte abundance. Overall, our findings suggest that TPHP may have multiple mechanisms of toxicity leading to an increase in ionocyte abundance and pericardial edema within developing zebrafish embryos., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Previous studies have shown that altered expression of a family of small noncoding RNAs (microRNAs, or miRs) regulates the expression of downstream mRNAs and is associated with diseases and developmental disorders. miR133b is highly expressed in mammalian cardiac and skeletal muscle, and aberrant expression is associated with cardiac disorders and electrophysiological changes in cardiomyocytes. Similarly, cardiac dysfunction has been observed in early life-stage mahi-mahi ( Coryphaena hippurus ) exposed to crude oil, a phenotype that has been associated with an upregulation of miR133b as well as subsequent downregulation of a delayed rectifier potassium channel (I Kr ) and calcium signaling genes that are important for proper heart development during embryogenesis. To examine the potential role of miR133b in oil-induced early life-stage cardiotoxicity in fish, cleavage-stage zebrafish ( Danio rerio ) embryos were either (1) microinjected with ∼3 nL of negative control miR (75 μM) or miR133b (75 μM) or (2) exposed to a treatment solution containing 5 μM benzo(a)pyrene (BaP), a model polycyclic aromatic hydrocarbon, as a positive control. At 72 h post fertilization (hpf), miR133b-injected fish exhibited BaP-like cardiovascular malformations, including a significantly increased pericardial area relative to negative control miR-injected embryos, as well as a significantly reduced eye area. qPCR revealed that miR133b microinjection decreased the abundance of cardiac-specific I Kr kcnh6 at 5 hpf, which may contribute to action potential elongation in oil-exposed cardiomyocytes. Additionally, ryanodine receptor 2, a crucial calcium receptor in the sarcoplasmic reticulum, was also downregulated by miR133b. These results indicate that an oil-induced increase in miR133b may contribute to cardiac abnormalities in oil-exposed fish by targeting cardiac-specific genes essential for proper heart development.

Dorsoventral (DV) patterning is a key landmark of embryonic development that is primarily regulated by bone morphogenetic protein (BMP) signaling. Disruption of DV patterning can result in downstream effects on cell specification and organogenesis. Zebrafish embryos have been extensively used to understand signaling pathways that regulate DV patterning because zebrafish embryos develop ex utero and, in contrast to mammalian embryos, which develop in utero, can be observed in real time using brightfield and fluorescence microscopy. Embryos with disrupted DV patterning are either dorsalized or ventralized, with lack of development of head or trunk/tail structures, respectively. Although these phenotypes are typically accompanied by effects on BMP signaling, exceptions exist where some drugs or environmental chemicals can disrupt DV patterning in the absence of effects on BMP signaling. Therefore, assessments of DV patterning should be accompanied by BMP signaling-specific readouts to confirm the role of BMP disruption. Here, we describe an exposure paradigm and steps for phenotyping zebrafish embryos for two types of DV defects, dorsalization and ventralization, with a range of severities. In addition, we describe a strategy for whole-mount immunohistochemistry of zebrafish embryos with an antibody specific for phospho-SMAD 1/5/9 (pSMAD 1/5/9), as disruption in pSMAD 1/5/9 localization is indicative of an effect on BMP signaling. Taken together, these protocols describe an initial strategy for evaluating DV patterning defects under various experimental conditions and confirming BMP-mediated DV patterning disruptions, which can be followed by additional studies that aim to uncover mechanisms leading to these adverse phenotypes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Phenotyping for dorsalization and ventralization Basic Protocol 2: Whole-mount immunohistochemistry with antibody to phospho-SMAD 1/5/9., (© 2021 Wiley Periodicals LLC.)

Polycyclic aromatic hydrocarbons (PAHs) are pervasive pollutants in aquatic ecosystems, and developing fish embryos are especially sensitive to PAH exposure. Exposure to crude oil or phenanthrene (a reference PAH found in oil) produces an array of gross morphological abnormalities in developing fish embryos, including cardiotoxicity. Recently, studies utilizing transcriptomic analyses in several oil-exposed fish embryos found significant changes in the abundance of transcripts involved in cholesterol biosynthesis. Given the vital role of cholesterol availability in embryonic heart development, we hypothesized that cholesterol dysregulation in early development contributes to phenanthrene-induced cardiotoxicity. We exposed zebrafish embryos to 12 or 15 µM phenanthrene from 6 to 72 h post fertilization (hpf) and demonstrated that, in conjunction with pericardial edema and bradycardia, several genes (fdft1 and hmgcra) in the cholesterol biosynthetic pathway were significantly altered. When embryos were pretreated with a cholesterol solution from 6 to 24 hpf followed by exposure to phenanthrene from 24 to 48 hpf, the effects of phenanthrene on heart rate were partially mitigated. Despite changes in gene expression, whole-mount in situ staining of cholesterol was not significantly affected in embryos exposed to phenanthrene ranging in stage from 24 to 72 hpf. However, the 2-dimensional yolk area was significantly increased with phenanthrene exposure at 72 hpf, suggesting that lipid transport from the yolk to the developing embryo was impaired. Environ Toxicol Chem 2021;40:1586-1595. © 2021 SETAC., (© 2021 SETAC.)

Exposure to oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) at critical developmental time-points in fish models impairs red blood cell concentrations in a regioselective manner, with 2-hydroxychrysene being more potent than 6-hydroxychrysene. To better characterize this phenomenon, embryos of Japanese medaka (Oryzias latipes) were exposed to 2- or 6-hydroxychrysene (0.5, 2, or 5 μM) from 4 h-post-fertilization (hpf) to 7 d-post-fertilization. Following exposure, hemoglobin concentrations were quantified by staining fixed embryos with o-dianisidine (a hemoglobin-specific dye) and stained embryos were imaged using brightfield microscopy. Exposure to 2-hydroxychrysene resulted in a concentration-dependent decrease in hemoglobin relative to vehicle-exposed embryos, while only the highest concentration of 6-hydroxychrysene resulted in a significant decrease in hemoglobin. All tested concentrations of 2-hydroxychrysene also caused significant mortality (12.2 % ± 2.94, 38.9 % ± 14.4, 85.6 % ± 11.3), whereas mortality was not observed following exposure to 6-hydroxychrysene. Therefore, treatment of embryos with 2-hydroxychrysene at various developmental stages and durations was subsequently conducted to identify key developmental landmarks that may be targeted by 2-hydroxychrysene. A sensitive window of developmental toxicity to 2-hydroxychrysene was found between 52-100 hpf, with a 24 h exposure to 10 μM 2-hydroxychrysene resulting in significant anemia and mortality. Since exposure to 2-hydroxychrysene from 52 to 100 hpf, a window that includes liver morphogenesis in medaka, resulted in the highest magnitude of toxicity, liver development and function may have a role in 2-hydroxychrysene developmental toxicity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Chemicals are listed on California's Proposition 65 (Prop 65) for their potential to cause cancer, birth defects or other reproductive harm, and certain chemicals from this list are often detected within interior vehicle dust and air. Therefore, this study examined the potential risk associated with five Prop 65-listed chemicals detected within vehicle interiors: benzene, formaldehyde, di (2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), and tris(1,3-dichloro-2-propyl)phosphate (TDCIPP). Exposure estimates based on time spent within a vehicle were derived from a meta-analysis of estimated concentrations from the literature. Regulatory levels established by the California Office of Environmental Health Hazard Assessment (OEHHA) were then used to generate percent reference doses (%RfDs) for chemical-specific daily doses as well as determine the probability of risk (exceedance probability) as a function of %RfD for each chemical-specific daily dose. Based on our meta-analysis, benzene and formaldehyde were detected in vehicle interior air whereas DEHP, DBP and TDCIPP were detected in vehicle interior dust. Benzene and formaldehyde were the only two chemicals with an estimated %RfD > 100 across any of the commute times. For commute times of 20 min or longer, the %RfD was > 100 for maximum exposures based on the "maximum allowable daily level" for benzene, and for 95th-percentile exposures based on the "no significant risk level" for benzene and formaldehyde. Furthermore, the probability of exceeding 100% RfD was highest for cancer risks associated with benzene, followed by cancer risks associated with formaldehyde and the risk of reproductive and developmental toxicity associated with benzene. Lastly, within the entire state of California, the percent of commuters with a 10% probability of exceeding cancer risk associated with benzene or formaldehyde exposure was 78% and 63%, respectively. Overall, our study raises concerns about the potential risk associated with inhalation of benzene and formaldehyde for people who spend a significant amount of time in their vehicles, an issue that is especially pertinent to traffic-congested areas where people have longer commutes., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that, upon activation by ligands, heterodimerizes with retinoid X receptor (RXR), binds to PPAR response elements (PPREs), and activates transcription of downstream genes. As PPARγ plays a central role in adipogenesis, fatty acid storage, and glucose metabolism, PPARγ-specific pharmaceuticals (e.g., thiazolidinediones) have been developed to treat Type II diabetes and obesity within human populations. However, to our knowledge, no prior studies have concurrently assessed the effects of PPARγ ligand exposure on genome-wide PPARγ binding as well as effects on the transcriptome and lipidome within human cells at biologically active, non-cytotoxic concentrations. In addition to quantifying concentration-dependent effects of ciglitazone (a reference PPARγ agonist) and GW 9662 (a reference PPARγ antagonist) on human hepatocarcinoma (HepG2) cell viability, PPARγ abundance in situ , and neutral lipids, HepG2 cells were exposed to either vehicle (0.1% DMSO), ciglitazone, or GW 9662 for up to 24 h, and then harvested for 1) chromatin immunoprecipitation-sequencing (ChIP-seq) to identify PPARγ-bound regions across the entire genome, 2) mRNA-sequencing (mRNA-seq) to identify potential impacts on the transcriptome, and 3) lipidomics to identify potential alterations in lipid profiles. Following exposure to ciglitazone and GW 9662, we found that PPARγ levels were not significantly different after 2-8 h of exposure. While ciglitazone and GW 9662 resulted in a concentration-dependent increase in neutral lipids, the magnitude and localization of PPARγ-bound regions across the genome (as identified by ChIP-seq) did not vary by treatment. However, mRNA-seq and lipidomics revealed that exposure of HepG2 cells to ciglitazone and GW 9662 resulted in significant, treatment-specific effects on the transcriptome and lipidome. Overall, our findings suggest that exposure of human cells to PPARγ ligands at biologically active, non-cytotoxic concentrations results in toxicity that may be driven by a combination of both PPARγ-dependent and PPARγ-independent mechanisms., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Boscalid is a persistent fungicide that is frequently detected in surface waters and may be neurotoxic to aquatic organisms. Herein, we evaluated the effects of environmentally relevant boscalid concentrations to zebrafish to explore its potentially neurotoxic mechanisms of effect. Behavioral responses (swimming, phototaxis, and predation), histopathology, transcriptomics, biochemical parameter analysis and gene expression of larval and adult zebrafish following boscalid treatment were assessed. We found that boscalid significantly inhibited the locomotor ability and phototactic response of larvae after an 8-d exposure, and altered the locomotor activity, predation trajectories and ability in adults after a 21-d exposure. It was noted that predation rates of zebrafish were significantly decreased by 30% and 100% after exposure to 0.1 and 1.0 mg/L boscalid, respectively. Adverse alterations in the cell differentiation of eyes and brain injury were also observed in both larvae and adults following boscalid exposure. The expression of genes related to neurodevelopment, neurotransmission, eye development, and visual function, in conjunction with RNA-Seq results, indicated that boscalid may impair visual phototransduction and nervous system processes in larval zebrafish. Conclusively, boscalid exposure may affect the neurobehavioral response of zebrafish by impairing proper visual and nervous system function., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Menthol is widely used in tobacco products. This study compared the effects of menthol on human bronchial epithelium using submerged cultures, a VITROCELL® cloud chamber that provides air liquid interface (ALI) exposure without solvents or heating, and a Cultex ALI system that delivers aerosol equivalent to that inhaled during vaping. In submerged culture, menthol significantly increased calcium influx and mitochondrial reactive oxygen species (ROS) via the TRPM8 receptor, responses that were inhibited by a TRPM8 antagonist. VITROCELL® cloud chamber exposure of BEAS-2B monolayers increased mitochondrial protein oxidation, expression of the antioxidant enzyme SOD2, activation of NF-κB, and secretion of inflammatory cytokines (IL-6 and IL-8). Proteomics data collected following ALI exposure of 3D EpiAirway tissue in the Cultex showed upregulation of NRF-2-mediated oxidative stress, oxidative phosphorylation, and IL-8 signaling. Across the three platforms, menthol adversely effected human bronchial epithelium in a manner that could lead to respiratory disease., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Organophosphate esters (OPEs) are a class of semi-volatile organic compounds (SVOCs) used as flame retardants, plasticizers, and anti-foaming agents. Due to stringent flammability standards in vehicles and the ability of OPEs to migrate out of end-use products, elevated concentrations of OPEs have been found in car dust samples around the world. As many residents of Southern California spend a significant amount of time in their vehicles, there is potential for increased exposure to OPEs associated with longer commute times. As approximately 70% of the University of California, Riverside's undergraduate population commutes, the objective of this study was to use silicone wristbands to monitor personal exposure to OPEs and determine if exposure was associated with commute time in a subset of these students. Participants were asked to wear wristbands for five continuous days and complete daily surveys about the amount of time spent commuting. Data were then used to calculate a participant-specific total commute score. Components of Firemaster 550 (triphenyl phosphate, or TPHP, and isopropylated triaryl phosphate isomers) and Firemaster 600 (TPHP and tert-butylated triaryl phosphate isomers) - both widely used commercial flame retardant formulations - were strongly correlated with other OPEs detected within participant wristbands. Moreover, the concentration of tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) was significantly correlated with the concentration of several Firemaster 500 components and tris(2-chloroisopropyl) phosphate (TCIPP). Finally, out of all OPEs measured, TDCIPP was significantly and positively correlated with total commute score, indicating that longer commutes are associated with increased human exposure to TDCIPP. Overall, our findings raise concerns about the potential for chronic TDCIPP exposure within vehicles and other forms of transportation, particularly within densely populated and traffic-congested areas such as Southern California., Competing Interests: Declaration of Competing Interest We declare that we have no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Per- and polyfluoroalkyl substances (PFASs) have been used for decades within industrial processes and consumer products, resulting in frequent detection within the environment. Using zebrafish embryos, we screened 38 PFASs for developmental toxicity and revealed that perfluorooctanesulfonamide (PFOSA) was the most potent developmental toxicant, resulting in elevated mortality and developmental abnormalities following exposure from 6 to 24 h post fertilization (hpf) and 6 to 72 hpf. PFOSA resulted in a concentration-dependent increase in mortality and abnormalities, with surviving embryos exhibiting a >12-h delay in development at 24 hpf. Exposures initiated at 0.75 hpf also resulted in a concentration-dependent delay in epiboly, although these effects were not driven by a specific sensitive window of development. We relied on mRNA-sequencing to identify the potential association of PFOSA-induced developmental delays with impacts on the embryonic transcriptome. Relative to stage-matched vehicle controls, these data revealed that pathways related to hepatotoxicity and lipid transport were disrupted in embryos exposed to PFOSA from 0.75 to 14 hpf and 0.75 to 24 hpf. Therefore, we measured liver area as well as neutral lipids in 128-hpf embryos exposed to vehicle (0.1% DMSO) or PFOSA from 0.75 to 24 hpf and clean water from 24 to 128 hpf, and showed that PFOSA exposure from 0.75 to 24 hpf resulted in a decrease in liver area and increase in yolk sac neutral lipids at 128 hpf. Overall, our findings show that early exposure to PFOSA adversely impacts embryogenesis, an effect that may lead to altered lipid transport and liver development., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Environmental monitoring has demonstrated widespread occurrence of the flame-retardant tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), raising concerns about the impact on aquatic life. Using 1 H NMR and GC-MS metabolomics and 20-day body length experiments, we have determined that exposure to TDCIPP affects Artemia franciscana. The LC 50 for a 48 h TDCIPP exposure was determined to be 37.1 ± 1.3 μM. Acute exposure (48 h) to 20.0 μM did not affect A. franciscana body length but did elicit a metabolic change. Chronic exposure to 0.50 μM TDCIPP caused decreased body length in A. franciscana exposed for 20 days and elicited a metabolic response. Principal component analysis revealed variance between acute and chronic exposure along PC1 (36.4%) and between control and TDCIPP along PC2 (17.4%). One-way ANOVA indicated that 19 metabolites were significantly affected by TDCIPP exposure; namely metabolites of the osmolyte class, including betaine, phosphocholine, gadusol, taurine, glycerol and trehalose - metabolites that are essential osmoprotectants in extremophile species. Other pathways that may be perturbed by TDCIPP exposure include one carbon, glycine, serine, threonine, and glycerophospholipid metabolism., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor that regulates lipid/glucose homeostasis and adipocyte differentiation. While the role of PPARγ in adipogenesis and diabetes has been extensively studied, little is known about PPARγ function during early embryonic development. Within zebrafish, maternally-loaded pparγ transcripts are present within the first 6 h post-fertilization (hpf), and de novo transcription of zygotic pparγ commences at ~48 hpf. Since maternal pparγ transcripts are elevated during a critical window of cell fate specification, the objective of this study was to test the hypothesis that PPARγ regulates gastrulation and dorsoventral patterning during zebrafish embryogenesis. To accomplish this objective, we relied on (1) ciglitazone as a potent PPARγ agonist and (2) a splice-blocking, pparγ-specific morpholino to knockdown pparγ. We found that initiation of ciglitazone-a potent human PPARγ agonist-exposure by 4 hpf resulted in concentration-dependent effects on dorsoventral patterning in the absence of epiboly defects during gastrulation, leading to ventralized embryos by 24 hpf. Interestingly, ciglitazone-induced ventralization was reversed by co-exposure with dorsomorphin, a bone morphogenetic protein signaling inhibitor that induces strong dorsalization within zebrafish embryos. Moreover, mRNA-sequencing revealed that lipid- and cholesterol-related processes were affected by exposure to ciglitazone. However, pparγ knockdown did not block ciglitazone-induced ventralization, suggesting that PPARγ is not required for dorsoventral patterning nor involved in ciglitazone-induced toxicity within zebrafish embryos. Our findings point to a novel, PPARγ-independent mechanism of action and phenotype following ciglitazone exposure during early embryonic development., Competing Interests: The authors declare that they have no competing interests., (© 2019 Cheng et al.)

Many coastal systems have been experiencing the effects of non-chemical and chemical anthropological stressors through respective increases in surface water temperatures and rainstorm-derived runoff events of pyrethroid pesticide movement into waterways such as the San Francisco Bay-Delta. Salmonid populations in the Bay-Delta have been dramatically declining in recent decades. Therefore, the aim of this study was to investigate the interactive effects of bifenthrin, a pyrethroid insecticide, and increasing water temperatures on targeted neuroendocrine and behavioral responses in Chinook salmon (Oncorhynchus tshawytscha) parr (10- month post-hatch). Parr were reared at 11 °C, 16.4 °C, or 19 °C for 14 days and, in the final 96 h of rearing, exposed to nominal concentrations of 0, 0.15, or 1.5 μg/L bifenthrin. A predatory avoidance Y-Maze behavioral assay was conducted immediately following exposures. Parr were presented a choice of clean or odorant zones, and locomotive behavior was recorded. Thyroid hormones (T3 and T4), estradiol, and testosterone were quantified within plasma using ELISAs, and the expression of brain hormone and dopamine receptor genes were also evaluated by qPCR. Brain dopamine levels were analyzed by LC/MS. No significant changes were observed in brain transcripts or plasma hormone concentrations with bifenthrin or increasing temperature. However, temperature did significantly lower brain dopamine levels in fish reared at 19 °C compared to 11 °C controls, but was unaltered by bifenthrin treatment. In contrast, parr reared at 11 °C and exposed to 1.5 μg/L bifenthrin spent significantly less time avoiding a predatory odorant compared to vehicle controls reared at 11 °C. The 16.4 °C and 1.5 μg/L-treated fish spent significantly more time in the neutral arm compared to the odorant and clean arms, as well as spending significantly less time in the clean arm compared to the 11 °C control fish. These results suggest that the interaction of temperature and bifenthrin may be adversely impacting predator-avoidance behavior, which may not be related to dopaminergic responses., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Triphenyl phosphate (TPHP) is a commonly used organophosphate flame retardant and plasticizer in the United States. Using zebrafish as a model, the overall objective of this study was to identify potential organs that might be targeted by TPHP during embryonic development. Based on mRNA-sequencing, TPHP exposure from 24 to 30 h post fertilization (hpf) and 24 to 48 hpf significantly affected the abundance of 305 and 274 transcripts, respectively, relative to vehicle (0.1% DMSO) controls. In addition to minor effects on cardiotoxicity- and nephrotoxicity-related pathways, ingenuity pathway analysis (IPA) of significantly affected transcripts within 30- and 48-hpf embryos revealed that hepatotoxicity-related pathways were strongly affected following exposure to TPHP-alone. Moreover, although pretreatment with fenretinide (a retinoic acid receptor agonist) mitigated TPHP-induced pericardial edema and liver enlargement at 72 and 128 hpf, respectively, IPA revealed that fenretinide was unable to block TPHP-induced effects on cardiotoxicity-, nephrotoxicity-, and hepatotoxicity-related pathways at 48 hpf, suggesting that TPHP-induced effects on the transcriptome were not associated with toxicity later in development. In addition, based on Oil Red O staining, we found that exposure to TPHP nearly abolished neutral lipids from the embryonic head and trunk and, based on metabolomics, significantly decreased the total abundance of metabolites-including betaine, a known osmoprotectant-at 48 and 72 hpf. Overall, our data suggest that, in addition to the heart, TPHP exposure during early development results in adverse effects on the liver, lipid utilization, and osmoregulation within embryonic zebrafish., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

Importance: No previous studies have shown that acute inhalation of thirdhand smoke (THS) activates stress and survival pathways in the human nasal epithelium., Objective: To evaluate gene expression in the nasal epithelium of nonsmoking women following acute inhalation of clean air and THS., Design, Setting, and Participants: Nasal epithelium samples were obtained from participants in a randomized clinical trial (2011-2015) on the health effects of inhaled THS. In a crossover design, participants were exposed, head only, to THS and to conditioned, filtered air in a laboratory setting. The order of exposures was randomized and exposures were separated by at least 21 days. Ribonucleic acid was obtained from a subset of 4 healthy, nonsmoking women., Exposures: By chance, women in the subset were randomized to receive clean air exposure first and THS exposure second. Exposures lasted 3 hours., Main Outcomes and Measures: Differentially expressed genes were identified using RNA sequencing with a false-discovery rate less than 0.1., Results: Participants were 4 healthy, nonsmoking women aged 27 to 49 years (mean [SD] age, 42 [10.2] years) with no chronic diseases. A total of 389 differentially expressed genes were identified in nasal epithelium exposed to THS, while only 2 genes, which were not studied further, were affected by clean air. Enriched gene ontology terms associated with stress-induced mitochondrial hyperfusion were identified, such as respiratory electron transport chain (q = 2.84 × 10-3) and mitochondrial inner membrane (q = 7.21 × 10-6). Reactome pathway analysis identified terms associated with upregulation of DNA repair mechanisms, such as nucleotide excision repair (q = 1.05 × 10-2). Enrichment analyses using ingenuity pathway analysis identified canonical pathways related to stress-induced mitochondrial hyperfusion (eg, increased oxidative phosphorylation) (P = .001), oxidative stress (eg, glutathione depletion phase II reactions) (P = .04), and cell survival (z score = 5.026)., Conclusions and Relevance: This study found that acute inhalation of THS caused cell stress that led to the activation of survival pathways. Some responses were consistent with stress-induced mitochondrial hyperfusion and similar to those demonstrated previously in vitro. These data may be valuable to physicians treating patients exposed to THS and may aid in formulating regulations for the remediation of THS-contaminated environments.

Diphenyl phosphate (DPHP) is an aryl phosphate ester (APE) used as an industrial catalyst and chemical additive and is the primary metabolite of flame retardant APEs, including triphenyl phosphate (TPHP). Minimal DPHP-specific toxicity studies have been published despite ubiquitous exposure within human populations following metabolism of TPHP and other APEs. Therefore, the objective of this study was to determine the potential for DPHP-induced toxicity during embryonic development. Using zebrafish as a model, we found that DPHP significantly increased the distance between the sinus venosus and bulbus arteriosis (SV-BA) at 72 h postfertilization (hpf) following initiation of exposure before and after cardiac looping. Interestingly, pretreatment with d-mannitol mitigated DPHP-induced effects on SV-BA length despite the absence of DPHP effects on pericardial area, suggesting that DPHP-induced cardiac defects are independent of pericardial edema formation. Using mRNA-sequencing, we found that DPHP disrupts pathways related to mitochondrial function and heme biosynthesis; indeed, DPHP significantly decreased hemoglobin levels in situ at 72 hpf following exposure from 24 to 72 hpf. Overall, our findings suggest that, similar to TPHP, DPHP impacts cardiac development, albeit the potency of DPHP is significantly less than TPHP within developing zebrafish.

Polycyclic aromatic hydrocarbons (PAHs) present in crude oil can cause global gene dysregulation and developmental impairment in fish. However, the mechanisms that alter gene regulation are not well understood. In this study, larval red drum ( Sciaenops ocellatus) were exposed to water accommodated fractions of source oil (6.8, 13.7, and 35.9 μg/L total PAHs) and weathered slick oil (4.7, 8.1, and 18.0 μg/L total PAHs) from the Deepwater Horizon (DWH) oil spill. The global mRNA-microRNA functional networks associated with the toxicity of DWH oil were explored by next-generation sequencing and in-depth bioinformatics analyses. Both source and slick oil significantly altered the expression of miR-18a, miR-27b, and miR-203a across all exposure concentrations. Consistent with the observed concentration-dependent morphological changes, the target mRNAs of these microRNAs were predominantly involved in neuro-cardio system development processes and associated key signaling pathways such as axonal guidance signaling, cAMP-response-element-binding protein signaling in neurons, calcium signaling, and nuclear-factor-of-activated T cells signaling in cardiac hypertrophy. The results indicated that the developmental toxicity of crude oil may result from the abnormal expression of microRNAs and associated target genes, especially for the nervous system. Moreover, we provide a case study for systematic toxicity evaluation leveraging mRNA-microRNA-seq data using nonmodel species.

Bifenthrin (BF) is a pyrethroid insecticide widely used in urban and agricultural applications. Previous studies in embryos of zebrafish have shown that BF can affect estradiol biosynthesis and the dopaminergic system. To examine the role of the estrogen receptor (ER) in the endocrine effects of BF, embryos were exposed for 96 h to a mixture of 0.15 and 1.5 μg/L BF and an ER agonist (17α-ethynylestradiol - EE2) at 0.09 μg/L. Transcripts related to estrogenic (vitellogenin VTG) and dopaminergic (tyrosine hydroxylase (TH), dopamine receptor 1 (DR1), monoamine oxidase (MAO), and catechol-O-methyltransferase b (COMTb)) signaling pathways were investigated by qRT-PCR. Dopamine (DA) and its metabolites (homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC)) were also measured. There was a significant increase in VTG, DR1, MAO and COMTb mRNA levels and HVA-DA ratios within all zebrafish embryos exposed to EE2, including EE2 alone, 0.15 μg/L BF + EE2 and 1.5 μg/L BF + EE2. A significant decrease in homogenate concentrations of DA was observed within all zebrafish embryos exposed to EE2, which included EE2 alone, 0.15 μg/L BF + EE2 and 1.5 μg/L BF + EE2. Co-exposure of BF with EE2 failed to diminish estrogenic or dopaminergic signaling in embryos. Additionally, embryos with diminished ERα expression by morpholino injection were exposed to 0.15 μg/L BF, 1.5 μg/L BF and 0.09 μg/L EE2, with subsequent gene expression measurements. ERα knockdown did not prevent the effects of BF, indicating ERα may have a limited role in the estrogenic and dopaminergic effects caused by BF in zebrafish embryos., (Copyright © 2018 Elsevier B.V. All rights reserved.)

There is a need for fast, efficient, and cost-effective hazard identification and characterization of chemical hazards. This need is generating increased interest in the use of zebrafish embryos as both a screening tool and an alternative to mammalian test methods. A Collaborative Workshop on Aquatic Models and 21st Century Toxicology identified the lack of appropriate and consistent testing protocols as a challenge to the broader application of the zebrafish embryo model. The National Toxicology Program established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative to address the lack of consistent testing guidelines and identify sources of variability for zebrafish-based assays. This report summarizes initial SEAZIT information-gathering efforts. Investigators in academic, government, and industry laboratories that routinely use zebrafish embryos for chemical toxicity testing were asked about their husbandry practices and standard protocols. Information was collected about protocol components including zebrafish strains, feed, system water, disease surveillance, embryo exposure conditions, and endpoints. Literature was reviewed to assess issues raised by the investigators. Interviews revealed substantial variability across design parameters, data collected, and analysis procedures. The presence of the chorion and renewal of exposure media (static versus static-renewal) were identified as design parameters that could potentially influence study outcomes and should be investigated further with studies to determine chemical uptake from treatment solution into embryos. The information gathered in this effort provides a basis for future SEAZIT activities to promote more consistent practices among researchers using zebrafish embryos for toxicity evaluation.

Niclosamide is an antihelminthic drug used worldwide for the treatment of tapeworm infections. Recent drug repurposing screens have revealed that niclosamide exhibits diverse mechanisms of action and, as a result, demonstrates promise for a number of 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, the objective of this study was to investigate the mechanism of niclosamide toxicity during early stages of embryonic development. Using zebrafish as a model, we found that niclosamide induced a concentration-dependent delay in epiboly progression during late-blastula and early-gastrula, an effect that was dependent on exposure during the maternal-to-zygotic transition-a period characterized by degradation of maternally derived transcripts, zygotic genome activation, and initiation of cell motility. Moreover, we found that niclosamide did not affect embryonic oxygen consumption, suggesting that oxidative phosphorylation-a well-established target for niclosamide within intestinal parasites-may not play a role in niclosamide-induced epiboly delay. However, mRNA-sequencing revealed that niclosamide exposure during blastula and early-gastrula significantly impacted the timing of zygotic genome activation as well as the abundance of cytoskeleton- and cell cycle regulation-specific transcripts. In addition, we found that niclosamide inhibited tubulin polymerization in vitro, suggesting that niclosamide-induced delays in epiboly progression may, in part, be driven by disruption of microtubule formation and cell motility within the developing embryo.

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.

The adverse outcome pathway (AOP) framework can be used to help support the development of alternative testing strategies aimed at predicting adverse outcomes caused by triggering specific toxicity pathways. In this paper, we present a case-study demonstrating the selection of alternative in chemico assays targeting the molecular initiating events of established AOPs, and evaluate use of the resulting data to predict higher level biological endpoints. Based on two AOPs linking inhibition of the deiodinase (DIO) enzymes to impaired posterior swim bladder inflation in fish, we used in chemico enzyme inhibition assays to measure the molecular initiating events for an array of 51 chemicals. Zebrafish embryos were then exposed to 14 compounds with different measured inhibition potentials. Effects on posterior swim bladder inflation, predicted based on the information captured by the AOPs, were evaluated. By linking the two datasets and setting thresholds, we were able to demonstrate that the in chemico dataset can be used to predict biological effects on posterior chamber inflation, with only two outliers out of the 14 tested compounds. Our results show how information organized using the AOP framework can be employed to develop or select alternative assays, and successfully forecast downstream key events along the AOP. In general, such in chemico assays could serve as a first-tier high-throughput system to screen and prioritize chemicals for subsequent acute and chronic fish testing, potentially reducing the need for long-term and costly toxicity tests requiring large numbers of animals., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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 coexposed embryos to HX 531-a pan-RXR antagonist-from 24 to 72 hpf and, contrary to our hypothesis, found that coexposure 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.

Bifenthrin is a pyrethroid insecticide used in urban and agricultural applications. Previous studies have shown that environmentally relevant (ng/L) concentrations of bifenthrin increased plasma concentrations of 17β-estradiol (E2) and altered the expression of dopaminergic pathway components. The dopaminergic neurons can indirectly regulate E2 biosynthesis, suggesting that bifenthrin may disrupt the hypothalamic-pituitary-gonadal (HPG) axis. Because embryos do not have a complete HPG axis, the hypothesis that bifenthrin impairs dopamine regulation was tested in embryonic and 1-mo-old juvenile zebrafish (Danio rerio) with exposure to measured concentrations of 0.34 and 3.1 µg/L bifenthrin for 96 h. Quantitative reverse transcriptase polymerase chain reaction was used to investigate transcripts of tyrosine hydroxylase (TH), dopamine receptor 1 (DR1) and 2A (DR2A), dopamine active transporter (DAT), estrogen receptor α (ERα), ERβ1, ERβ2, luteinizing hormone β (LHβ), follicle-stimulating hormone β (FSHβ), vitellogenin (VTG), cytochrome P450 cyp19a1a, and cyp19a1b. Levels of E2 were measured by enzyme-linked immunosorbent assay (ELISA). Dopamine and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) concentrations were measured by liquid chromatrography-tandem mass spectrometry (LC-MS/MS). Significant decreases in TH and DR1 transcripts and HVA levels, as well as ratios of HVA/dopamine and HVA+DOPAC/dopamine, in zebrafish embryos were observed after bifenthrin treatment. In juveniles, a significant increase in the expression of ERβ1 and the DOPAC to dopamine ratio was noted. These results show a possible antiestrogenic effect of bifenthrin in embryos, and estrogenicity in juveniles, indicating life-stage-dependent toxicity in developing fish. Environ Toxicol Chem 2018;37:236-246. © 2017 SETAC., (© 2017 SETAC.)

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a high-production volume organophosphate flame retardant widely used within the United States. Within zebrafish, initiation of TDCIPP exposure at 0.75 h post-fertilization (hpf) results in genome-wide alterations in methylation during cleavage (2 hpf) as well as epiboly delay or arrest (at higher concentrations) during late-blastula and early-gastrula (4-6 hpf). To determine whether these TDCIPP-induced effects were associated with impacts on the transcriptome, embryos were exposed to vehicle (0.1% DMSO) or 2 µM TDCIPP from 0.75 hpf to 6 hpf, and total RNA was extracted from triplicate embryo pools per treatment and hybridized onto duplicate Affymetrix Zebrafish Gene 1.0 ST Arrays per RNA sample. Based on transcriptome-wide profiling, TDCIPP resulted in a significant impact on biological processes involved in dorsoventral patterning and bone morphogenetic protein (BMP) signaling. Consistent with these responses, TDCIPP exposure also resulted in strongly dorsalized embryos by 24 hpf-a phenotype that mimicked the effects of dorsomorphin, a potent and selective BMP inhibitor. Moreover, the majority of dorsalized embryos were preceded by epiboly arrest at 6 hpf. Our microarray data also revealed that the expression of sizzled ( szl )-a gene encoding a secreted Frizzled-related protein that limits BMP signaling-was significantly decreased by nearly 4-fold at 6 hpf. Therefore, we used a splice-blocking morpholino to test the hypothesis that knockdown of szl phenocopies TDCIPP-induced delays in epiboly progression. Interestingly, contrary to our hypothesis, injection of szl MOs did not affect epiboly progression but, similar to chordin ( chd ) morphants, resulted in mildly ventralized embryos by 24 hpf. Overall, our findings suggest that TDCIPP-induced epiboly delay may not be driven by decreased szl expression, and that TDCIPP-induced dorsalization may-similar to dorsomorphin-be due to interference with BMP signaling during early zebrafish development., Competing Interests: The authors declare there are no competing interests.

The impact of unmonitored contaminants, also known as contaminants of emerging concern (CECs), on freshwater streams remains largely uncharacterized. Water samples from 31 streams representing urban, agricultural and undeveloped (i.e., open space) land use in Southern California (USA) were analyzed for in vitro and in vivo bioactivity. The extent and magnitude of bioactivity screened using endocrine-responsive cell bioassays and a fish embryo screening assay were low. In contrast, a wider gradient of responses for the aryl hydrocarbon receptor (AhR) assay was observed, which was negatively correlated with a measure of benthic community structure. Both aromatic and non-aromatic CECs were tentatively identified in these samples, but polycyclic aromatic hydrocarbons (PAHs), known AhR agonists in urban environments, were not present at detectable levels. These results suggest that a combination of in vitro and in vivo show potential as screening techniques for biological condition in situ, but that more advanced, comprehensive analytical methods are needed to identify bioactive contaminants.

Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) is an organophosphate flame retardant that impacts zebrafish epiboly - an effect that may be associated with genome-wide hypomethylation. Using zebrafish as a model, the objectives of this study were to (1) quantify concentration-dependent impacts of TDCIPP on epiboly; (2) determine whether co-exposure with folic acid (FA) - a methyl donor - mitigates TDCIPP-induced impacts; and (3) using ten previously identified TDCIPP-susceptible loci, rely on bisulfite amplicon sequencing (BSAS) to monitor CpG methylation dynamics across multiple TDCIPP concentrations in the presence or absence of FA. Embryos were exposed to TDCIPP from 0.75 h post-fertilization (hpf) to 2, 4, 6, or 24 hpf in the presence or absence of 1 mM FA. Although TDCIPP delayed epiboly up to 3 h by 6 hpf and induced malformations by 24 hpf, FA was unable to mitigate TDCIPP-induced effects at all stages evaluated. Moreover, while no differences in global methylation were detected using a 5-methylcytosine (5-mC) DNA ELISA, BSAS revealed that TDCIPP-induced effects on CpG methylation were dependent on concentration and developmental stage, and that early effects on methylation do not persist despite continuous exposure. Our findings demonstrate that TDCIPP delays zebrafish epiboly, a phenotype that is preceded by complex, dynamic alterations in DNA methylation., Competing Interests: Conflict of Interest Statement The authors declare no conflict of interest.

Spontaneous activity represents an early, primitive form of motor activity within zebrafish embryos, providing a potential readout for identification of neuroactive compounds. However, despite use as an endpoint in chemical screens around the world, the predictive power and limitations of assays relying on spontaneous activity remain unclear. Using an improved high-content screening assay that increased throughput from 384 to 3072 wells per week, we screened a well-characterized library of 1280 pharmacologically active compounds (LOPAC 1280 ) - 612 of which target neurotransmission - to identify which targets are detected using spontaneous activity as a readout. Results from this screen revealed that (1) 8% of the LOPAC 1280 library was biologically active; (2) spontaneous activity was affected by compounds spanning a broad array of targets; (3) only 4% of compounds targeting neurotransmission impacted spontaneous activity; and (4) hypoactivity was observed for 100% of hits detected, including those that exhibit opposing mechanisms of action for the same target. Therefore, while this assay was able to rapidly identify potent neuroactive chemicals, these data suggest that spontaneous activity may lack the ability to discriminate modes of action for compounds interfering with neurotransmission, an issue that may be due to systemic uptake following waterborne exposure, persistent control variation, and/or interference with non-neurotransmission-related mechanisms., (Copyright © 2017 Elsevier Inc. All rights reserved.)

One of the primary sources of polycyclic aromatic hydrocarbons (PAHs) in marine environments is oil. Photochemical oxidation and microbial transformation of PAH-containing oils can result in the formation of oxygenated products. Among the PAHs in crude oil, chrysene is one of the most persistent within the water column and may be transformed to 2- and 6-hydroxychrysene (OHCHR). Both of these compounds have been shown to activate (2-OHCHR) and antagonize (6-OHCHR) the estrogen receptor (ER). Previous studies in our lab have shown that estrogen can significantly alter zebrafish development. However, little is known about the developmental toxicity of hydroxylated PAHs. Zebrafish embryos were exposed to 0.5-10μM of 2- or 6-OHCHR from 2h post-fertilization (hpf) until 76hpf. A significant decrease in survival was observed following exposure to 6-OHCHR - but not 2-OHCHR. Both OHCHRs significantly increased the percentage of overall deformities after treatment. In addition to cardiac malformations, ocular and circulatory defects were also observed in embryos exposed to both compounds, while 2-OHCHR generally resulted in a higher prevalence of effect. Moreover, treatment with 2-OHCHR resulted in a significant decrease in hemoglobin levels. ER nor G-Protein coupled estrogen receptor (GPER) antagonists and agonists did not rescue the observed defects. We also analyzed the expression of cardiac-, eye- and circulation-related genes previously shown to be affected by oil. Rhodopsin mRNA expresssion was significantly decreased by both compounds equally. However, exposure to 2-OHCHR significantly increased the expression of the hematopoietic regulator, runx1 (runt related transcription factor 1). These results indicate the toxicity of oxygenated photoproducts of PAHs and suggest that other targets and signaling pathways may contribute to developmental toxicity of weathered oil. Our findings also demonstrate the regio-selective toxicity of hydroxy-PAHs in the effects on eye and circulatory development and raise the need to identify mechanisms and ecological risks of oxy-PAHs to fish populations., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Exposure to 17β-estradiol (E2) influences the regulation of multiple signaling pathways, and E2-mediated disruption of signaling events during early development can lead to malformations such as cardiac defects. In this study, we investigated the potential role of the G-protein estrogen receptor 1 (GPER) in E2-induced developmental toxicity. Zebrafish embryos were exposed to E2 from 2h post fertilization (hpf) to 76 hpf with subsequent transcriptional measurements of heart and neural crest derivatives expressed 2 (hand2), leucine rich repeat containing 10 (lrrc10), and gper at 12, 28 and 76 hpf. Alteration in the expression of lrrc10, hand2 and gper was observed at 12 hpf and 76 hpf, but not at 28 hpf. Expression of these genes was also altered after exposure to G1 (a GPER agonist) at 76 hpf. Expression of lrrc10, hand2 and gper all coincided with the formation of cardiac edema at 76 hpf as well as other developmental abnormalities. While co-exposure of G1 with G36 (a GPER antagonist) rescued G1-induced abnormalities and altered gene expression, co-exposure of E2 with G36, or ICI 182,780 (an estrogen receptor antagonist) did not rescue E2-induced cardiac deformities or gene expression. In addition, no effects on the concentrations of downstream ER and GPER signaling molecules (cAMP or calcium) were observed in embryo homogenates after E2 treatment. These data suggest that the impacts of E2 on embryonic development at this stage are complex and may involve multiple receptor and/or signaling pathways., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) is a high-production volume organophosphate-based plasticizer and flame retardant widely used within the United States. Using zebrafish as a model, the objectives of this study were to determine whether (1) TDCIPP inhibits DNA methyltransferase (DNMT) within embryonic nuclear extracts; (2) uptake of TDCIPP from 0.75 h postfertilization (hpf, 2-cell) to 2 hpf (64-cell) or 6 hpf (shield stage) leads to impacts on the early embryonic DNA methylome; and (3) TDCIPP-induced impacts on cytosine methylation are localized to CpG islands within intergenic regions. Within this study, 5-azacytidine (5-azaC, a DNMT inhibitor) was used as a positive control. Although 5-azaC significantly inhibited zebrafish DNMT, TDCIPP did not affect DNMT activity in vitro at concentrations as high as 500 μM. However, rapid embryonic uptake of 5-azaC and TDCIPP from 0.75 to 2 hpf resulted in chemical- and chromosome-specific alterations in cytosine methylation at 2 hpf. Moreover, TDCIPP exposure predominantly resulted in hypomethylation of positions outside of CpG islands and within intragenic (exon) regions of the zebrafish genome. Overall, these findings provide the foundation for monitoring DNA methylation dynamics within zebrafish as well as identifying potential associations among TDCIPP exposure, adverse health outcomes, and DNA methylation status within human populations.

Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPOX), an enzyme that catalyzes oxidation of protoporphyrinogen IX to protoporphyrin IX during chlorophyll and heme biosynthesis. Based on a high-content screen, we previously identified butafenacil as a potent inducer of anemia in zebrafish embryos. Therefore, the objective of this study was to begin investigating the utility of butafenacil as a positive control for identifying anemia- and variegate porphyria-inducing chemicals. Static exposure to butafenacil from 5 to 72 h post-fertilization (hpf) in glass beakers resulted in a concentration-dependent decrease in arterial circulation at low micromolar concentrations. At 72 hpf, the magnitude of butafenacil-induced anemia was similar when embryos were exposed in the presence or absence of light, whereas protoporphyrin accumulation and acute toxicity were significantly lower or absent when embryos were exposed under dark conditions. To identify sensitive developmental windows, we treated embryos to butafenacil from 5, 10, 24, or 48 hpf to 72 hpf in the presence of light, and found that anemia and protoporphyrin accumulation were present at 72 hpf following initiation of exposure at 5 and 10 hpf. On the contrary, protoporphyrin accumulation - but not anemia - was present following initiation of exposure at 24 hpf. Lastly, protoporphyrin accumulation at 72 hpf after exposure from 24 to 48 hpf suggests that protoporphyrin was not eliminated over a 24-h recovery period. Collectively, our data suggests that butafenacil may be a reliable positive control for identifying anemia- and variegate porphyria-inducing chemicals.

During early zebrafish embryogenesis, spontaneous tail contractions represent the first sign of locomotion and result from innervation of primary motoneuron axons to target axial muscles. Based on a high-content screen, we previously demonstrated that exposure of zebrafish embryos to abamectin--an avermectin insecticide--from 5-25 hours post-fertilization (hpf) abolished spontaneous activity in the absence of effects on survival and gross morphology. Therefore, the objective of this study was to begin investigating the mechanism of abamectin-induced hypoactivity in zebrafish. Similar to 384-well plates, static exposure of embryos to abamectin from 5-25 hpf in glass beakers resulted in elimination of activity at low micromolar concentrations. However, abamectin did not affect neurite outgrowth from spinal motoneurons and, compared with exposure from 5-25 hpf, embryos were equally susceptible to abamectin-induced hypoactivity when exposures were initiated at 10 and 23 hpf. Moreover, immersion of abamectin-exposed embryos in clean water resulted in complete recovery of spontaneous activity relative to vehicle controls, suggesting that abamectin reversibly activated ligand-gated chloride channels and inhibited neurotransmission. To test this hypothesis, we pretreated embryos to vehicle or non-toxic concentrations of fipronil or endosulfan--two insecticides that antagonize the γ-aminobutyric acid (GABA) receptor--from 5-23 hpf, and then exposed embryos to vehicle or abamectin from 23-25 hpf. Interestingly, activity levels within abamectin-exposed embryos pretreated with either antagonist were similar to embryos exposed to vehicle alone. Using quantitative PCR and phylogenetic analyses, we then confirmed the presence of GABA receptor α1 and β2 subunits at 5, 10, and 23 hpf, and demonstrated that zebrafish GABA receptor subunits are homologous to mammalian GABA receptor subunits. Overall, our data collectively suggest that abamectin induces rapid and reversible hypoactivity within early zebrafish embryos, an effect that may be mediated through the GABA receptor., (Copyright © 2015. Published by Elsevier Inc.)

Using zebrafish as a model, we previously reported that developmental exposure to triphenyl phosphate (TPP) - a high-production volume organophosphate-based flame retardant - results in dioxin-like cardiac looping impairments that are independent of the aryl hydrocarbon receptor. Using a pharmacologic approach, the objective of this study was to investigate the potential role of retinoic acid receptor (RAR) - a nuclear receptor that regulates vertebrate heart morphogenesis - in mediating TPP-induced developmental toxicity in zebrafish. We first revealed that static exposure of zebrafish from 5-72h post-fertilization (hpf) to TPP in the presence of non-toxic concentrations of an RAR antagonist (BMS493) significantly enhanced TPP-induced toxicity (relative to TPP alone), even though identical non-toxic BMS493 concentrations mitigated retinoic acid (RA)-induced toxicity. BMS493-mediated enhancement of TPP toxicity was not a result of differential TPP uptake or metabolism, as internal embryonic doses of TPP and diphenyl phosphate (DPP) - a primary TPP metabolite - were not different in the presence or absence of BMS493. Using real-time PCR, we then quantified the relative change in expression of cytochrome P450 26a1 (cyp26a1) - a major target gene for RA-induced RAR activation in zebrafish - and found that RA and TPP exposure resulted in a ∼5-fold increase and decrease in cyp26a1 expression, respectively, relative to vehicle-exposed embryos. To address whether TPP may interact with human RARs, we then exposed Chinese hamster ovary cells stably transfected with chimeric human RARα-, RARβ-, or RARγ to TPP in the presence of RA, and found that TPP significantly inhibited RA-induced luciferase activity in a concentration-dependent manner. Overall, our findings suggest that zebrafish RARs may be involved in mediating TPP-induced developmental toxicity, a mechanism of action that may have relevance to humans., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Firemaster 550 (FM550) is an additive flame retardant mixture used within polyurethane foam and is increasingly found in house dust and the environment due to leaching. Despite the widespread use of FM550, very few studies have investigated the potential toxicity of its ingredients during early vertebrate development. In the current study, we sought to specifically investigate mono-substituted isopropylated triaryl phosphate (mITP), a component comprising approximately 32% of FM550, which has been shown to cause cardiotoxicity during zebrafish embryogenesis. Previous research showed that developmental defects are rescued using an aryl hydrocarbon receptor (AHR) antagonist (CH223191), suggesting that mITP-induced toxicity was AHR-dependent. As zebrafish have three known AHR isoforms, we used a functional AHR2 knockout line along with AHR1A- and AHR1B-specific morpholinos to determine which AHR isoform, if any, mediates mITP-induced cardiotoxicity. As in silico structural homology modeling predicted that mITP may bind favorably to both AHR2 and AHR1B isoforms, we evaluated AHR involvement in vivo by measuring CYP1A mRNA and protein expression following exposure to mITP in the presence or absence of CH223191 or AHR-specific morpholinos. Based on these studies, we found that mITP interacts with both AHR2 and AHR1B isoforms to induce CYP1A expression. However, while CH223191 blocked mITP-induced CYP1A induction and cardiotoxicity, knockdown of all three AHR isoforms failed to block mITP-induced cardiotoxicity in the absence of detectable CYP1A induction. Overall, these results suggest that, while mITP is an AHR agonist, mITP causes AHR-independent cardiotoxicity through a pathway that is also antagonized by CH223191., (Copyright © 2014 Elsevier B.V. All rights reserved.)