Tris(1,3-dichloro-2-propyl) Phosphate Exposure During the Early-Blastula Stage Alters the Normal Trajectory of Zebrafish Embryogenesis

Dasgupta, Subham; Cheng, Vanessa; Vliet, Sara M.F.; Mitchell, Constance A.; Volz, David C.

doi:10.1021/acs.est.8b03730

Cited by 63 publications

(32 citation statements)

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“…Phenotyping for DV defects has been previously used for the detection of early developmental toxicity in large-scale screens for drugs or small molecules that target developmental signaling pathways. Specifically, because BMP signaling primarily regulates DV patterning, studies from our lab and other groups have used dorsalization and ventralization phenotypes as indicators of BMP signaling defects caused by BMP inhibitors such as DMP, activators such as 4 -H (Dasgupta et al, 2018;Vrijens et al, 2013;Yu et al, 2008), or knockdown of BMP pathway-specific genes such as szl and chd (Dasgupta et al, 2017). However, our work also showed that DV defects are not necessarily readouts of direct impacts on BMP signaling because two structurally diverse chemicals, namely tris(1,3-dichloro-2propyl)phosphate (TDCIPP, an environmental chemical) and ciglitazone (a drug), induced dorsalization and ventralization phenotypes, respectively, without corresponding impacts on BMP signaling based on mRNA sequencing and pSMAD 1/5/9 staining (Cheng et al, 2019;Dasgupta et al, 2018;Dasgupta et al, 2017).…”

Section: Commentary Background Informationmentioning

confidence: 99%

Utilizing Zebrafish Embryos to Reveal Disruptions in Dorsoventral Patterning

2021

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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

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Section: Commentary Background Informationmentioning

confidence: 99%

Utilizing Zebrafish Embryos to Reveal Disruptions in Dorsoventral Patterning

2021

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“…This was further supported by our mRNA-seq data, as neither bmp2 nor psmad1/5/9 transcripts were significantly altered after exposure to 12.5 μM ciglitazone, 0.078 μM DMP, or 12.5 μM ciglitazone + 0.078 μM DMP. Similarly, exposure to tris(1,3-dichloro-2-propyl) phosphate (TDCIPP)—a high-production volume organohalogen flame retardant—induced dorsoventral patterning defects in the absence of effects on BMP signaling within developing zebrafish embryos (Dasgupta et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

“…Maximum tolerated concentrations for DMP and ciglitazone co-exposures were optimized based on preliminary experiments that tested combinations of 0.078 or 1.56 μM DMP in the presence or absence of 9.375, 12.5, or 15 μM ciglitazone. Although 0.625 µM DMP was used in our prior studies (Dasgupta et al, 2017; Dasgupta et al, 2018), we relied on 0.078 µM DMP since co-exposure with 0.625 μM DMP and 12.5 μM ciglitazone resulted in a significant increase in mortality. At 8 hpf, embryos were fixed overnight in 4% PFA/1× PBS.…”

Section: Methodsmentioning

confidence: 99%

“…At 8 hpf, embryos were fixed overnight in 4% PFA/1× PBS. Fixed embryos were manually dechorionated and then incubated overnight with anti-phosphoSMAD 1/5/9 IgG antibody (1:100 dilution; Cell Signaling Technology, Danvers, MA, USA) using previously described protocols (Yozzo, McGee & Volz, 2013; Dasgupta et al, 2018). Embryos were then incubated overnight with an IgG-specific Alexa Fluor-conjugated secondary antibody (1:500 dilution; Millipore Sigma, St. Louis, MO, USA).…”

Section: Methodsmentioning

confidence: 99%

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Ciglitazone—a human PPARγ agonist—disrupts dorsoventral patterning in zebrafish

Cheng¹,

Dasgupta²,

Reddam³

et al. 2019

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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.

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“…to be transferred to offspring, resulting in developmental neurotoxicity (Du et al, 2015;Liu et al, 2013;Yu et al, 2017;Zhang et al, 2018a;Zhang et al, 2018b). Additional adverse effects of OPE exposure include developmental toxicity (Dasgupta et al, 2018;Dasgupta et al, 2017;Jacobsen et al, 2017;Li et al, 2018;Oliveri et al, 2018;Yan & Hales, 2018), oxidative stress (Chen et al, 2015a;Chen et al, 2015b;Gu et al, 2019;Yan et al, 2017), thyroid disruption (Fernie et al, 2017;Hill et al, 2018;Liu et al, 2019;Preston et al, 2017), neurotoxicity (Andresen et al, 2004;Hong et al, 2018;Shi et al, 2018;Slotkin et al, 2017), carcinogenicity (Hoffman et al, 2017b;Ni et al, 2007;Van den Eede et al, 2013b), endocrine disruption (Arukwe et al, 2016;Kojima et al, 2013;Kojima et al, 2016;, and cellular toxicity (Shen et al, 2019;Canbaz et al, 2017;Crump et al, 2012;Shen et al, 2019;Su et al, 2014a agonists, similar to their parent compound TBOEP as well as TEHP, and TNBP (Kojima et al, 2016). Metabolism can significantly influence the accumulation and adverse effects of chemicals in organisms, however the transformation of OPEs and distribution of their metabolites are still unknown.…”

Section: Metabolismmentioning

confidence: 99%

Metabolism and fate of organophosphate ester (OPE) contaminants in rainbow trout (Oncorhynchus mykiss) using in vitro lab-based studies

Large¹

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Due to ubiquitous use of organophosphate esters (OPEs) as flame retardants and plasticizers, they have been found at high concentrations in a variety of environmental media while having low or non-detectable levels in biota, including fish. The exposure and fate of OPEs in fish depends on understanding the toxicokinetics, though studies on OPE metabolism are lacking. A model in vitro microsomal-based metabolism assay for rainbow trout (Oncorhynchus mykiss) was utilized to investigate OPE metabolism. Metabolic depletion was only noted for 2 alkyl OPEs and formation of corresponding diester metabolites was low, indicating other unidentified metabolites were formed. Comparison with avian and mammalian models showed fish generally had slower and less complete metabolism, illustrating species-specific differences in biotransformation. Structure also influenced OPE metabolism in that bulky, aryl OPEs were not metabolized. These results provide important information of toxicokinetic processes that can affect the exposure and fate of OPEs in rainbow trout. Chapter 1: General Introduction 1.1 Flame Retardant Chemicals and Organophosphate Esters In today's homes and public spaces, flammability concerns are well warranted. Increased use of plastics, foams, synthetic fiber-base fillings, and electronics bring an increased risk of fire as many of these polymers can be highly flammable. Industry flammability standards, such as California's Technical Bulletin 117, were implemented in an effort to reduce fires and increase average escape time (US EPA, 2007) and, as a result, production and use of flame retardants has become common practice to meet flammability standards in various consumer goods, textiles, furniture, electronics, and plastics.

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Tris(1,3-dichloro-2-propyl) Phosphate Exposure During the Early-Blastula Stage Alters the Normal Trajectory of Zebrafish Embryogenesis

Cited by 63 publications

References 51 publications

Utilizing Zebrafish Embryos to Reveal Disruptions in Dorsoventral Patterning

Utilizing Zebrafish Embryos to Reveal Disruptions in Dorsoventral Patterning

Ciglitazone—a human PPARγ agonist—disrupts dorsoventral patterning in zebrafish

Metabolism and fate of organophosphate ester (OPE) contaminants in rainbow trout (Oncorhynchus mykiss) using in vitro lab-based studies

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