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Disruption of erythropoiesis by dioxin in the zebrafish
Developmental Dynamics
Abstract
2,3,7,8-Tetrachlorodibenzo-p- dioxin (TCDD, or dioxin) causes early life stage mortality in a variety of fish species. We have used the zebrafish (Danio rerio) to study the cardiovascular effects of TCDD treatment over the time course of zebrafish development. Early TCDD exposure (6 ng/ml) starting at 4 hr postfertilization (hpf) produced reductions in blood flow and in the number of circulating erythrocytes. These defects were consistently observable by 72 hpf. However, these responses were not observed when TCDD exposure was delayed until 96 hpf or later. These results suggest a model in which TCDD interferes with cardiovascular and erythropoietic developmental processes that are normally completed by 96 hpf. This model is strengthened by the finding that TCDD exposure blocks the step in hematopoiesis in which developing zebrafish switch from the primitive phase to the definitive phase of erythropoiesis. We observed no effect of TCDD on the levels of circulating primitive erythrocytes before 72 hpf and the expression of markers for early hematopoiesis, GATA-1 and GATA-2. However, early TCDD exposure prevented the appearance of definitive phase erythrocytes. TCDD produced a small delay in the migration of blood cells expressing SCL from the intermediate cell mass to the dorsal mesentery and dorsal aorta. Despite the decrease in blood flow produced by TCDD, confocal microscopy of the trunk vasculature by using a Tie2/green fluorescence protein endothelial marker at 48, 60, 72, and 96 hpf of TCDD-exposed (4 hpf) revealed no apparent defects in blood vessel structure. © 2001 Wiley-Liss, Inc.
... In addition, pericardial area showed a dose-response trend, with the 4 and 8 mg DCA/L groups having significantly larger pericardial areas than the 1 and 2 mg DCA/L groups. Pericardial edema, indicated by increased pericardial area, is frequently observed among embryos and eleutheroembryos exposed to a wide variety of contaminants with diverse modes of action, including pharmaceuticals (Carlsson and Norrgren 2004;Akande et al. 2010), TCDD (Henry et al. 1997;Belair et al. 2001;Hill 2004;Antkiewicz et al. 2005), PAHs (Colavecchia et al. 2006;Ana dos Anjos et al. 2011;He et al. 2012), and heavy metals (Cheng et al. 2000;Zhu et al. 2014;Chen et al. 2015). ...
... Of these, gata-1 and bmp10 did not quantify in any of the samples (cycle threshold >34), indicating that their expression in whole larvae is unlikely to be a suitable FET test endpoint. It is possible that using isolated tissues or in situ hybridization would improve detection as expression of gata-1 and bmp10 is localized to the intermediate cell mass and heart, respectively (Detrich et al. 1995;Amatruda and Zon 1999;Laux et al. 2013), and others have had success quantifying gata-1 expression using in situ hybridization (Belair et al. 2001;Cui et al. 2016). In contrast to gata-1 and bmp10, my17 was detectable in all samples, yet there were no significant differences in its expression between groups ( Figure 10; ANOVA, p ¼ 0.39). ...
... Pericardial area was not only responsive to chemical exposure, but has been seen after exposure to chemicals with Mass À À À À Snout-vent length þ þ þ þ Growth-related gene expression igf À À À À ghr À À À À Spontaneous contraction frequency À À À À Eye size index À À þ þ Neurologically related gene expression shh þ À À À gfap þ À À À Heart rate À À À þ Pericardial area þ þ þ þ Cardiovascular system-related gene expression myl7 À À À À vegfa À À þ þ À ¼ no significant difference from control; þ ¼ significant difference from control. gfap ¼ glial fibrillary acidic protein; ghr ¼ growth hormone receptor; igf ¼ insulin-like growth factor; my17 ¼ atrial light chain 2; shh ¼ sonic hedgehog; vegfa ¼ vascular endothelial growth factor A. different modes of action (Henry et al. 1997;Cheng et al. 2000;Belair et al. 2001;Carlsson and Norrgren 2004;Hill 2004;Antkiewicz et al. 2005;Colavecchia et al. 2006;Akande et al. 2010;Ana dos Anjos et al. 2011;He et al. 2012;Chen et al. 2015). Furthermore, it has been linked to adverse biological effects (Hicken et al. 2011), making it a promising candidate as a sublethal endpoint for the FET test. ...
The fathead minnow fish embryo toxicity (FET) test has been proposed as a more humane alternative to current toxicity testing methods, as younger organisms are thought to experience less distress during toxicant exposure. However, the FET test protocol does not include endpoints that allow for the prediction of sublethal adverse outcomes, limiting its utility relative to other test types. Researchers have proposed the development of sublethal endpoints for the FET test to increase its utility. The present study 1) developed methods for previously unmeasured sublethal metrics in fathead minnows (i.e., spontaneous contraction frequency and heart rate) and 2) investigated the responsiveness of several sublethal endpoints related to growth (wet weight, length, and growth‐related gene expression), neurodevelopment (spontaneous contraction frequency, and neurodevelopmental gene expression), and cardiovascular function and development (pericardial area, eye size and cardiovascular related gene expression) as additional FET test metrics using the model toxicant 3,4–dichloroaniline. Of the growth, neurological and cardiovascular endpoints measured, length, eye size and pericardial area were found to more responsive than the other endpoints, respectively. Future studies linking alterations in these endpoints to longer‐term adverse impacts are needed to fully evaluate the predictive power of these metrics in chemical and whole effluent toxicity testing. This article is protected by copyright. All rights reserved
... Zebrafish eggs were statically exposed in water to either TCDD (1 ng/ml) or vehicle (DMSO) in 5 ml glass scintillation vials for 1 h with rocking (Belair et al., 2001). After exposure, fish were rinsed in TCDD-free water and placed in housing aquaria until analysis. ...
... This increased the variability in flow in the TCDD-exposed group. A higher dose of TCDD has been shown to completely halt circulation by 96 hpf (Belair et al., 2001;Henry et al., 1997); however, we chose the dose to be consistent with our extensive collection of embryo cardiotoxicity data at this level. ...
... However, this is not evidence of an unusual response. TCDD produces the same response as seen with the caAHR at 5 ng/ml, only fivefold higher than our standard 1 ng/ml dose (Belair et al., 2001;Henry et al., 1997). Continuous exposure to PCB-126 also produces an AHR2-dependent phenotype in zebrafish that occurs earlier and is more severe than what we observe with this TCDD exposure protocol (Grimes et al., 2008). ...
Exposure of zebrafish embryos to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activates the zebrafish aryl hydrocarbon receptor 2 (AHR) to produce developmental and cardiovascular toxicity.
AHR is found in the heart; however, AHR activation by TCDD is not confined to the heart and occurs throughout the organism.
In order to understand the cause of cardiotoxicity, we constructed a constitutively active AHR (caAHR) based on the zebrafish
AHR2 and expressed it specifically in cardiomyocytes. We show that AHR activation within the cardiomyocytes can account for
the heart failure induced by TCDD. Expression of the caAHR within the heart produced cardiac malformations, loss of circulation,
and pericardial edema. The heart-specific activation of AHR reproduced several other well-characterized endpoints of TCDD
toxicity outside of the cardiovascular system, including defects in swim bladder and craniofacial development. This work identifies
a single cellular site of TCDD action, the myocardial cell, that can account for the severe cardiovascular collapse observed
following early life stage exposure to TCDD, and contributes to other forms of toxicity.
... Aryl hydrocarbon receptor is conserved throughout the vertebrate lineage, including zebrafish (Hahn et al., 1997). Zebrafish embryos are sensitive to TCDD exposure, exhibiting severe pericardial edema (PCE), abnormal heart looping, circulation failure, disrupted erythropoiesis, and reduced jaw length (Belair et al., 2001;Henry et al., 1997;Teraoka et al., 2002). In contrast to mice, zebrafish have 3 paralogs of AHR: ahr1a, ahr1b, and ahr2 (Andreasen, Hahn, et al., 2002;Karchner et al., 2005;Tanguay et al., 1999). ...
... 1-way ANOVA). We next compared jaw length between ahr1 mutant and wildtype larvae, and did not observe a significant difference compared with wild type in single or double ahr1 mutants ( Ahr2, But Not Ahr1a or Ahr1b, Is Required for TCDDinduced Cardiotoxicity in Embryos 2,3,7,8-tetrachlorodibenzo-p-dioxin is a persistent environmental toxin that acts as a carcinogen in humans and causes cardiotoxicity and mortality at nanomolar concentrations in zebrafish embryos (Belair et al., 2001;Flesch-Janys et al., 1995;Henry et al., 1997). Ahr2 is known to mediate the toxic effects of TCDD (Garcia et al., 2018;Goodale et al., 2012;Tanguay et al., 1999), yet it is not clear whether zebrafish ahr1 genes are also required for TCDD toxicity. ...
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that binds environmental toxicants and regulates gene expression. AHR also regulates developmental processes, like craniofacial development and hematopoiesis, in the absence of environmental exposures. Zebrafish have three paralogues of AHR: ahr1a, ahr1b and ahr2. Adult zebrafish with mutations in ahr2 exhibited craniofacial and fin defects. However, the degree to which ahr1a and ahr1b influence ahr2 signaling and contribute to fin and craniofacial development are not known. We compared morphology of adult ahr2 mutants and ahr1a;ahr1b single and double mutant zebrafish. We found that ahr1a;ahr1b single and double mutants were morphologically normal while ahr2 mutant zebrafish demonstrated fin and craniofacial malformations. At 5 days post fertilization, both ahr1a;ahr1b and ahr2 mutant larvae were normal, suggesting that adult phenotypes are due to defects in maturation or maintenance. Next, we analyzed the function of zebrafish AHRs activated by environmental ligands. The prototypical AHR ligand, TCDD, induces toxicity in humans and rodents via AHR and causes cardiotoxicity in zebrafish embryos. It has been shown that embryos with mutations in ahr2 are resistant to TCDD toxicity, yet it is unclear whether ahr1 receptors are required. Further, though AHR was shown to interact with estrogen receptor alpha following TCDD treatment, it is not known whether this interaction is constitutive or context-dependent. To determine whether estrogen receptors are constitutive cofactors for AHR signaling, we used genetic and pharmacologic techniques to analyze TCDD-dependent toxicity in estrogen receptor and ahr mutant embryos. We found that embryos with mutations in ahr1a;ahr1b or estrogen receptor genes are susceptible to TCDD toxicity while ahr2 mutant embryos are TCDD-resistant. Moreover, pharmacologic blockade of nuclear estrogen receptors failed to prevent TCDD toxicity. These findings suggest that ahr1 genes do not have overlapping functions with ahr2 in fin and craniofacial development or TCDD-dependent toxicity, and that estrogen receptors are not constitutive partners of ahr2.
... Hormis un retard de croissance, la réponse embryotoxique aux dioxines la plus fréquente rencontrée chez les poissons est l'oedème péricardiaque et/ou du sac vitellin, causé par une perturbation des fonctions circulatoires et osmorégulatrices, des malformations cardiovasculaires ou encore une augmentation de la perméabilité vasculaire,également responsable d'hémorragies (Belair et al., 2001;Henry et al., 1997;Hill et al., 2004a,b;Teraoka et al., 2002;. Les composés apparentés aux dioxines affectentégalement la chondrogenèse dans son ensemble, cela se traduisant par une atrophie parfois prononcée de la mâchoire inférieure, bien que la croissance de l'ensemble du cartilage crânien soit altérée (Hill et al., 2004a;Hornung et al., 1999;Kim et Cooper, 1999;Teraoka et al., 2006). ...
... These dioxinlike compounds (DLCs) are persistent and widespread pollutants that accumulate in fish tissues due to their lipophilic properties. Responses of early life stages of fish to PHHs include induction of cytochrome P4501A (CYP1A) detoxification enzymes, developmental arrest, craniofacial deformities, pericardial and yolk sac edemas, hemorrhages and mortality (Belair et al., 2001;Henry et al., 1997;Hill et al., 2004a,b;Teraoka et al., 2002). Lethal embryotoxicity of PHHs may have contributed to lake trout (Salvelinus namaycush) decline in Lake Ontario, Canada, between 1940and 1980(Cook et al., 2003. ...
... Pericardial edema and yolk sac edema are hallmark signs of TCDD developmental toxicity in fish, chick and mammalian species (Brunstrom and Lund, 1988;Gilbertson et al., 1991;Guiney et al., 2000;Henry et al., 1997;Matsumura et al., 1997;Rifkind et al., 1985). Pericardial edema is the distinctive sign of TCDD-induced toxicity and appears from at 72 hpf in zebrafish (Belair et al., 2001;Dong et al., 2002;Henry et al., 1997;Hill et al., 2004). In this study, pericardial edema in crucian carp exposed to TCDD started at 78 hpf and especially the area of pericardial edema showed a significant increase compared to DMSO-exposed control group (Fig. 5). ...
... Yolk sac edema was first observed at 78 hpf and showed a significant increase at later time (at 102 hpf) than pericardial edema (at 78 hpf). This timing of onset is consistent with other reports (Belair et al., 2001;Dong et al., 2002;Henry et al., 1997;Hill et al., 2004;Wisk and Cooper, 1992). The ED50 for yolk sac edema in crucian carp was calculated as 0.06 ng TCDD/g embryo (ED50) at 126 hpf. ...
... The zebrafish (Danio rerio) has been used as a model for studying the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Henry et al., 1997). By using zebrafish, it has been possible to determine that TCDD exposure during development causes heart failure and circulation collapse (Belair et al., 2001;Antkiewicz et al., 2005;Heideman et al., 2005). Interestingly, it is only during heart development that fish are sensitive to TCDD cardiotoxic effects: TCDD does not appear to harm the juvenile or adult heart (Lanham et al., 2012;Hofsteen et al., 2013). ...
... At 96 hpf, we found that loss of sox9b caused an approximately 2fold decrease in the RBC perfusion rate relative to wild-type controls (Table 1). However, the decrease in RBC perfusion rate of sox9b null larvae did not approach the complete halt of RBC movement in the tail of TCDD-exposed larvae (Belair et al., 2001;Antkiewicz et al., 2005). ...
Activation of the transcription factor AHR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prevents the formation of the epicardium and leads to severe heart malformations in developing zebrafish (Danio rerio). The downstream genes that cause heart malformation are not known. Because TCDD causes craniofacial malformations in zebrafish by downregulating the sox9b gene, we hypothesized that cardiotoxicity might also result from sox9b downregulation. We found that sox9b is expressed in the developing zebrafish heart ventricle and that TCDD exposure markedly reduces this expression. Furthermore, we found that manipulation of sox9b expression could phenocopy many but not all of the effects of TCDD at the heart. Loss of sox9b prevented the formation of epicardium progenitors comprising the proepicardium on the pericardial wall, and prevented the formation and migration of the epicardial layer around the heart. Zebrafish lacking sox9b showed pericardial edema, an elongated heart and reduced blood circulation. Fish lacking sox9b failed to form valve cushions and leaflets. Sox9b is one of two mammalian Sox9 homologs, sox9b and sox9a. Knock down of sox9a expression did not cause cardiac malformations, or defects in epicardium development. We conclude that the decrease in sox9b expression in the heart caused by TCDD plays a role in many of the observed signs of cardiotoxicity. We find that while sox9b is expressed in myocardial cells, it is not normally expressed in the affected epicardial cells or progenitors. We therefore speculate that sox9b is involved in signals between the cardiomyocytes and the nascent epicardial cells.
... TCDD exposure produces decreased cardiac output, reduced end diastolic volume, and decreased peripheral blood flow. This ultimately leads to heart failure, which includes ventricular standstill and total loss of circulation Belair et al., 2001;Henry et al., 1997). One of the most interesting features of TCDD-induced cardiotoxicity is the fact that zebrafish carrying a lethal body burden of TCDD develop normal appearing hearts through heart tube formation and looping, with a functional circulation. ...
... In zebrafish, an established model for studying cardiovascular development and disease, TCDD exposure results in valve malformation (Mehta et al., 2008), reduced heart size , and impaired development of the bulbus arteriosus (Grimes et al., 2008;Mehta et al., 2008). TCDD-exposed zebrafish larvae have decreased cardiac output, reduced end diastolic volume, and decreased peripheral blood flow Belair et al., 2001;Carney et al., 2006). Heart failure steadily worsens to ventricular standstill and total loss of circulation . ...
Embryonic exposure to the environmental contaminant and aryl hydrocarbon receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), disrupts cardiac development and function in fish, birds, and mammals. In zebrafish, the temporal
window of sensitivity to the cardiotoxic effects of TCDD coincides with epicardium formation. We hypothesized that this TCDD-induced
heart failure results from disruption of epicardial development. To determine whether embryonic TCDD exposure inhibits epicardium
and proepicardium (PE) development in zebrafish, we used histology and fluorescence immunocytochemistry to examine the epicardium
formation in fish exposed to TCDD. TCDD exposure prevented epicardium formation. Using live imaging and in situ hybridization, we found that TCDD exposure blocked the formation of the PE cluster. In situ hybridization experiments showed that TCDD exposure also prevented the expression of the PE marker tcf21 at the site where the PE normally forms. TCDD also inhibited expansion of the epicardial layer across the developing heart:
Exposure after PE formation was completed prevented further expansion of the epicardium. However, TCDD exposure did not affect
epicardial cells already present. Because TCDD blocks epicardium formation, but is not directly toxic to the epicardium once
complete, we propose that inhibition of epicardium formation can account for the window of sensitivity to TCDD cardiotoxicity
in developing zebrafish. Epicardium development is crucial to heart development. Loss of this layer during development may
account for most if not all of the TCDD-induced cardiotoxicity in zebrafish.
... Similar alterations in erythropoiesis, resembling signs of anemia resulting from cytotoxic damage to red blood cells, have been observed with other toxicants such as cadmium [64], copper nanoparticles [65], dioxin [66], and nitrite [14]. ...
Simple Summary
Bisphenol A (BPA) is an organic compound used in the manufacture of plastic products and is considered a common pollutant in the aquatic environment. Its impact on aquatic organisms is manifested in several reactions, particularly in changes in peripheral blood and disorders of hematopoiesis. In the present study, the effects of high concentrations of BPA on several indices of zebrafish blood were investigated. Differences in the effects of high and low concentrations on blood composition and disorders in hematopoietic organs were identified. The genotoxic effect of BPA at concentrations of 6 and 8 mg/L was confirmed. The test parameters most affected by the toxicant, such as the number of young erythrocytes in peripheral blood, nuclear anomalies of erythrocytes, and disturbances in hematopoiesis processes in the head kidney, expressed as changes in the ratio of immature/undifferentiated cell elements, were determined.
Abstract
In this study, the results of evaluating the acute toxicity of Bisphenol A on Danio rerio are presented, encompassing peripheral blood parameters, the composition of hematopoietic cells of erythroid and myeloid lines in the head kidney, and data from histological studies. The LC50 values of Bisphenol A for adult zebrafish individuals for 12, 24, and 48–96 h were determined, which were 18.04, 7.55, and 6.22 mg/L, respectively. The study includes data on the morphology and quantitative frequency of specific cells in the hematopoietic tissue of the head kidney, along with the consideration of adaptive mechanisms in hematopoiesis under BPA exposure. The application of polynomial regression analysis to reveal the concentration–effect relationship for some hematological and histological parameters was demonstrated. Significant increases in the frequency of erythrocyte nuclear abnormalities were observed at BPA concentrations of 6 and 8 mg/L, which indicates a genotoxic effect. BPA’s impact on fish peripheral blood parameters manifested as an increase in the number of erythrocytes (RBC) and immature erythrocytes, as well as a decrease in the number of lymphocytes. The most notable pathological changes in the head kidney’s hematopoietic tissue included circulatory disturbances and the formation of inflammation/degradation foci, as confirmed by histopathologic indices. At BPA concentrations of 2 and 4 mg/L, the observed changes were compensated for by hematopoietic adaptation mechanisms; however, at concentrations of 6 and 8 mg/L, acute systemic toxicity was evident.
... In MEP cells, chronic AHR activation causes differentiation of erythroid cells, and repression of AHR activity leads to megakaryocyte specification (Smith et al., 2013). However, several reports show that exposure of zebrafish (Belair et al., 2001;Carney et al., 2006) and human (Sakamoto et al., 2004) erythrocytes to the AHR agonist 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) damaged the deformation ability of erythrocytes and decreased hemoglobin synthesis. Thus, the role of AHR in the development and differentiation of erythroid cells is still ambiguous. ...
The aryl hydrocarbon receptor (AHR) plays an important role during mammalian embryo development. Inhibition of AHR signaling promotes the development of hematopoietic stem/progenitor cells. AHR also regulates the functional maturation of blood cells, such as T cells and megakaryocytes. However, little is known about the role of AHR modulation during the development of erythroid cells. In this study, we used the AHR antagonist StemRegenin 1 (SR1) and the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during different stages of human erythropoiesis to elucidate the function of AHR. We found that antagonizing AHR signaling improved the production of human embryonic stem cell (hESC)-derived erythrocytes and enhanced erythroid terminal differentiation. RNA-sequencing showed that SR1 treatment of proerythroblasts upregulated the expression of erythrocyte differentiation-related genes and downregulated actin organization-associated genes. We found that SR1 accelerated F-actin remodeling in terminally differentiated erythrocytes, favoring their maturation of the cytoskeleton and enucleation. We demonstrated that the effects of AHR inhibition on erythroid maturation were associated with F-actin remodeling. Our findings help uncover the mechanism for AHR-mediated human erythroid cell differentiation. We also provide a new approach toward the large-scale production of functionally mature human pluripotent stem cell-derived erythrocytes for use in translational applications.
... The AhR is a transcription factor, which after activation by DLCs translocates to the nucleus and alters the transcription of numerous genes. Fish are especially sensitive to DLCs: symptoms of DLCs toxicity have been described in various species and include the induction of cytochrome P4501A (CYP1A) detoxification enzymes, developmental arrest, cardiovascular defects, craniofacial deformities, pericardial and yolk sac edemas and hemorrhages (Belair et al., 2001;Henry et al., 1997;Teraoka et al., 2002). These are part of the so-called blue sac disease (BSD) syndrome, which can lead to death when severe enough. ...
Salmonids are known to be among the most sensitive fish to dioxin-like compounds (DLCs), but very little is known about the sensitivity of the brown trout (Salmo trutta), which has declined and is endangered in several countries of Europe and Western Asia. We investigated the sensitivity of brown trout larvae to a widespread dioxin-like PAH, retene (3.2 to 320 μg.L−1), compared to the larvae of a salmonid commonly used in toxicology studies, the rainbow trout (Oncorhynchus mykiss). Mortality, growth, cyp1a induction and the occurrence of deformities were measured after 15 days of exposure. Brown trout larvae showed a significantly higher mortality at 320 μg.L−1 compared to rainbow trout larvae. While the occurrence of deformities was only significantly increased at 320 μg.L−1 for the rainbow trout, brown trout larvae displayed pericardial edemas and hemorrhages already at 10 or 100 μg.L−1. cyp1a induction was increased significantly already at ≥3.2 μg.L−1 for the brown trout, versus ≥32 μg.L−1 for the rainbow trout. Least square regression analysis of the concentration-response relationships suggested that S. trutta larvae were at least 2 times more sensitive than O. mykiss larvae for cyp1a induction. The present study suggests that S. trutta larvae are more sensitive than O. mykiss larvae to a potent DLC, retene. As it is possible that S. trutta populations have declined partly because of pollution by DLCs, we recommend generating more data regarding the sensitivity of threatened fish populations, in order to ensure better risk assessment.
... In a different study, it was demonstrated that exposure to TCDD (a strong Ahr agonist), altered erythropoiesis with a decrease in blood flow, and this effect did not translate to observable defects in blood vessel structure in zebrafish fry (Antkiewicz et al., 2005;Belair et al., 2001;Carney et al., 2006). Overall, our data showing that fuel oils modulated ahr1b, vegfaa and ahrr expressions, however, associated alterations of phenotypic outcomes related to craniofacial and cardiovascular deformations are not directly comparable, probably due to the complex chemical composition of the fuel oil types (see below). ...
Due to the heavy fuel oil (HFO) ban in Arctic maritime transport and new legislations restricting the sulphur content of fuel oils, new fuel oil types are continuously developed. However, the potential impacts of these new fuel oil types on marine ecosystems during accidental spills are largely unknown. In this study, we studied the toxicity of three marine fuel oils (two marine gas oils with low sulphur contents and a heavy fuel oil) in early life stages of cod (Gadus morhua). Embryos were exposed for 4 days to water-soluble fractions of fuel oils at concentrations ranging from 4.1 - 128.3 µg TPAH/L, followed by recovery in clean seawater until 17 days post fertilization. Exposure to all three fuel oils resulted in developmental toxicity, including severe morphological changes, deformations and cardiotoxicity. To assess underlying molecular mechanisms, we studied fuel oil-mediated activation of aryl hydrocarbon receptor (Ahr) gene battery and genes related to cardiovascular, angiogenesis and osteogenesis pathways. Overall, our results suggest comparable mechanisms of toxicity for the three fuel oils. All fuel oils caused concentration-dependant increases of cyp1a mRNA which paralleled ahrr, but not ahr1b transcript expression. On the angiogenesis and osteogenesis pathways, fuel oils produced concentration-specific transcriptional effects that were either increasing or decreasing, compared to control embryos. Based on the observed toxic responses, toxicity threshold values were estimated for individual endpoints to assess the most sensitive molecular and physiological effects, suggesting that unresolved petrogenic components may be significant contributors to the observed toxicity.
... AhRs are found in high concentrations in the liver (Doering et al., 2014) and other AhR ligands have been shown to cause anemia. Belair et al. (2001) reported that 2,3,7,8-tetrachlorodibenzodioxin (TCDD), a potent AhR agonist, disrupted erythropoiesis in zebrafish embryos. Anemia was only found if zebrafish were exposed to TCDD before 96 hpf but after 48 hpf which also corresponds to liver development. ...
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 52-100 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.
... The embryo that survived in the aqueous methanol extract at the concentration of 300 mg/mL was positive for yolk sac oedema and bent tail. Yolk sac oedema in zebrafish might be due to the impairment of the blood flow or fluid (Belair et al., 2001). The bent tail observed can be associated with the ability in interfering BMP proteins and its pathway to suppress tumour metastasis (Rusdi et al., 2019). ...
Purpose
Moringa oleifera (MO) is a herbal remedy that is rich in essential amino acids and phytochemicals in the leaves, seeds and pods. It is becoming a famous food in various ethnic groups, and the consumption is increasing worldwide. However, there is no safety profile of the hydroalcoholic MO leaf extracts, or documented teratogenicity aspect. This study was sought to compare the antioxidant activity and the teratogenicity of hydroalcoholic MO leaf extracts in the zebrafish embryo.
Design/methodology/approach
MO leaves were extracted with aqueous alcohols (ethanol or methanol: water, 80:20 v/v) using the Soxhlet method. The antioxidant capacity was determined using DPPH assay. The concentrations of extracts ranging from 3 to 1,000 µg/mL were used to examine the toxicity and teratogenicity on the zebrafish embryo.
Findings
Both hydroalcoholic MO leaf extracts were positive for alkaloids, terpenoids, steroids, tannins, flavonoids, phenols, coumarins, quinones and glycosides, except saponins only absent in aqueous methanol extract. The antioxidant capacity based on lC 50 , was 15.92 ± 3.62 mg/mL for aqueous methanolic and 25.28 ± 2.89 mg/mL for aqueous ethanolic extract. For acute toxicity, the aqueous methanolic extract has lower LC 50 (163.87 ± 12.88 µg/mL) compared to aqueous ethanolic extract (337.48 ± 30.04 µg/mL). The embryo treated with aqueous methanolic extract showed phenotypic defect but not on the aqueous ethanolic extract.
Originality/value
This study suggests that aqueous methanol of MO leaves extract has better antioxidant capacity compared to ethanol, and ethanolic leaf extract is safer (higher LC50 and no teratogenicity) than methanolic extract.
... It is possible that the frequency of feeding PCB 1254 by Zhang and Qiao (2004) was too low and hence the damage was repaired between PCB exposures compared with our study of continuous daily exposure for 28 days. The results of our study are consistent with the findings of Belair et al. (2001), which reported that, the number of RBCs decreased in zebrafish exposed to 6 ng/mL 2,3,7,8-TCDD. A significant reduction in the number of red blood cells in this study may be due to hemolysis and damage to the erythrocyte membrane. ...
Dioxin-like polychlorinated biphenyls (DLPCBs) are ubiquitous environmental contaminants spread all over the world. They can cause disorders in the reproductive, nervous, gut, and immune systems. We investigated the effects of DL-PCB extracted from Zhanjiang (Guangdong Province, China) offshore area on the immune functions of adult zebrafish. Zebrafish were exposed to different levels of DL-PCBs, i.e., control, positive control (PCB77 at 16.0 μg/L), low (LD; PCB81 + PCB118 at 0.32 μg/L), and high-dose (HD; PCB81 + PCB118 at 16.0 μg/L) groups for 28 days. Compared with the control group, positive control and HD group showed a significant decrease (P < 0.05) in the number of red blood cells (RBC) on day 7 and the same decrease was observed in the LD group (P < 0.05) on day 21. The results of white blood cell (WBC) profiles were opposite to that of RBCs. Moreover, the serum lysozyme activity was significantly lower in positive control and HD group (P < 0.05) on day 21 but no significant effect was observed in the LD group. The mucus lysozyme activity and immunoglobulin concentration in positive control and HD group decreased significantly (P < 0.05) from day 14. A similar effect was observed in the LD group but was significant (P < 0.05) only on day 28. Additionally, histopathological examination showed accumulation of hemosiderin in the spleen of experimental animals, which was significant in positive control and HD group. Further, renal tubular epithelial cells of head kidney were swollen in the positive control and HD group while the expansion of lumen and renal interstitial edema was observed in the LD group on day 21 and with significant presence on 28 days. Therefore, these findings suggest that the exposure of zebrafish to DL-PCBs at > 16.0 μg/L can impair their immune functions.
... This indicates that the seasonality of red cell production is partially controlled by reproductive cycles and hormones (Pottinger and Pickering, 1987). Among environmental toxicants dioxin (Belair et al., 2001), the herbicide diuron (Reddy et al., 1992), cadmium (Dangre et al., 2010;Houston and Keen, 1984) and silver nanoparticles (Cui et al., 2016) have been shown to affect erythropoiesis. For the dioxin effect a reasonable explanation is the competition of hypoxia-inducible factor α and aryl hydrocarbon receptor for the same dimerization partner (Hofer et al., 2004). ...
The blood of fish has a continuous age distribution of erythrocytes. The properties of young and old erythrocytes differ with young erythrocytes being functionally much more versatile than old erythrocytes, which have higher haemoglobin content. Factors which affect the formation and breakdown of erythrocytes are reviewed. Erythropoiesis in fish is largely similar to that in mammals. However, definitive erythrocytes are mainly formed in the anterior part of the kidney, and erythropoietin is secreted mainly from the heart. Senescence of erythrocytes in fish has not been studied in detail, and consecutively the factors causing aging of erythrocytes in man are discussed. A major factor causing aging of erythrocytes is oxidative stress, which is also a major effect of toxicants on fish erythrocytes. Consequently, apparent age distribution of circulating erythrocytes will be affected by environmental pollution. At present, it is completely unknown, if the changes in age distribution of erythrocytes play a role in pollutant responses or if the age distribution of erythrocytes is actively regulated during environmental adaptation.
... The yolk sac edema observed might be due to B A the effects of the hemostasis, impaired blood flow or fluid imbalance. This postulation is supported by a study claiming the impaired blood flow and fluid imbalance is related to pericardial and yolk sac edema in zebrafish [15]. A study by Mishra and Tandon demonstrated that the water extracts derived from the leaves of a close species, B. spectabilis affected the hematologic cell count and packed cell volume in mice [16], which further suggests the possibility of Bougainvillea species in affecting the circulatory system. ...
Background: Bougainvillea glabra, or paper flower, is an ornamental plant known to possess various pharmacological activities. However, there is little information on the potential toxicity of this plant. Therefore, the aim of this study was to assess the acute toxicity and the potential teratogenic activity of water extracts of B. glabra bracts on zebrafish embryos which were selected as the model in this study.
Methods: The pink, purple, and dark pink bracts of B. glabra were extracted using the Soxhlet method. Embryos with the same division stage were selected and treated with the extracts (1-300 µg/mL) for 72 h. The mortality of the embryos was recorded and the teratogenicity effect induced by the extracts was identified. The yolk sac area and pigmentation were quantified using Image-J. The data were analyzed by IBM SPSS 22.0.
Results: All extracts within the tested concentrations did not induce death in embryo, except the pink bract extract with LC50 of 85.51 µg/mL. In the teratogenicity study, all the extract-treated embryos showed yolk sac edema at different concentrations, and the defect observed was independent of the concentrations. On top of that, the purple bract extract induced hypopigmentation in embryo, significantly at 30 µg/mL compared to control.
Conclusion: This study concluded that the water extracts derived from the pink, purple, and dark pink bracts of B. glabra have mild toxicity toward embryo.
... Upon exposure to a strong Ahr2 ligand, a toxicity phenotype that occurs early in zebrafish development is disruption of blood cell development and hemorrhaging (Belair et al. 2001;Henry et al. 1997). To elucidate the phenotypic impact of slincR expression on TCDD-induced hemorrhaging, we developmentally exposed control and slincR morphants to TCDD and recorded the presence or absence of hemorrhaging. ...
Background:
A structurally diverse group of chemicals, including dioxins [e.g., 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)] and polycyclic aromatic hydrocarbons (PAHs), can xenobiotically activate the aryl hydrocarbon receptor (AHR) and contribute to adverse health effects in humans and wildlife. In the zebrafish model, repression of sox9b has a causal role in several AHR-mediated toxic responses, including craniofacial cartilage malformations; however, the mechanism of sox9b repression remains unknown. We previously identified a long noncoding RNA, sox9b long intergenic noncoding RNA ( slincR), which is increased (in an AHR-dependent manner) by multiple AHR ligands and is required for the AHR-activated repression of sox9b.
Objective:
Using the zebrafish model, we aimed to enhance our understanding of the signaling events downstream of AHR activation that contribute to toxic responses by identifying: a) whether slincR is enriched on the sox9b locus, b) slincR's functional contributions to TCDD-induced toxicity, c) PAHs that increase slincR expression, and d) mammalian orthologs of slincR.
Methods:
We used capture hybridization analysis of RNA targets (CHART), qRT-PCR, RNA sequencing, morphometric analysis of cartilage structures, and hemorrhaging screens.
Results:
The slincR transcript was enriched at the 5' untranslated region (UTR) of the sox9b locus. Transcriptome profiling and human ortholog analyses identified processes related to skeletal and cartilage development unique to TCDD-exposed controls, and angiogenesis and vasculature development unique to TCDD-exposed zebrafish that were injected with a splice-blocking morpholino targeting slincR. In comparison to TCDD exposed control morphants, slincR morphants exposed to TCDD resulted in abnormal cartilage structures and a smaller percentage of animals displaying the hemorrhaging phenotype. In addition, slincR expression was significantly increased in six out of the sixteen PAHs we screened.
Conclusion:
Our study establishes that in zebrafish, slincR is recruited to the sox9b 5' UTR to repress transcription, can regulate cartilage development, has a causal role in the TCDD-induced hemorrhaging phenotype, and is up-regulated by multiple environmentally relevant PAHs. These findings have important implications for understanding the ligand-specific mechanisms of AHR-mediated toxicity. https://doi.org/10.1289/EHP3281.
... In this study, more red blood cells (RBCs) nearby the heart in BDE-47 treated larvae than control group indicated the interference of BDE-47 with erythropoiesis. TCDD was reported to interfere with cardiovascular system in developing zebrafish by producing a decrease in blood flow and a small delay in the migration of RBCs from the intermediate cell mass to the dorsal mesentery and dorsal aorta [42]. ...
Polybrominated diphenyl ethers (PBDEs) were once widely used as flame retardants in furniture and electronic products, and contamination persists in developing countries due to the dismantling of electronic waste. Our previous study confirmed that 2,2′,4,4′,5-pentabromodiphenyl ether (BDE-99) induced cytochrome P450 1A (Cyp1a) via aryl hydrocarbon receptor (Ahr)–mediated signaling in the zebrafish liver cell line (ZFL) in vitro. In this study, the toxicities of BDE-47 and BDE-99 at environmentally relevant concentrations (50 and 500 nM) were evaluated in newly hatched zebrafish (Danio rerio) larvae in vivo. A time-course study (8, 24, 48, and 96 h) was performed. BDE-99 was observed to cause yolk sac edema and pericardial edema after 72 h of exposure. Real-time polymerase chain reaction assay and whole-mount in situ hybridization assay confirmed cyp1a induction by BDE-99 in the liver and intestine. Continuous down-regulation of trβ by as much as 2.1-fold after 96 h and transient down-regulation of ttr by 7.1-fold after 24 h indicated the interference of BDE-99 in the thyroid hormone system. cyp1a induction was also observed in BDE-47–treated larvae, but cellular localization of cyp1a was not confirmed by whole-mount in situ hybridization. The induction of four cyp1 genes (cyp1a, cyp1b1, cyp1c1 and cyp1c2) by both BDE congeners warrants further study to understand the in vivo metabolism of BDE-47 and BDE-99 and the dioxin-like toxicity potencies of the OH-/MeO-PBDEs. The data obtained in this study will aid the characterization of molecular disorders caused by PBDEs in fish and help to delineate better models for toxicity assessment of environmental pollutants in ecological systems and in other vertebrates such as humans.
... This transparent vertebrate offers numerous compelling experimental advantages for evaluating toxic effects not found in other model systems (Sarmah and Marrs, 2016). A number of laboratories have employed the zebrafish to investigate the toxic effects of model chemicals released into the environment on the development of cardiovascular system, including nanoparticles, pesticides and various organic pollutants (Asharani et al., 2011;Belair et al., 2001;Chakraborty et al., 2016;Gerlach et al., 2014;Henry et al., 1997;King-Heiden et al., 2012;Teraoka et al., 2002;McGee et al., 2013;Zhang et al., 2013). These studies highlighted the suitability of the zebrafish as an excellent model to study human cardiovascular diseases (Bakkers, 2011). ...
... Studies have shown that the compounds that induced pericardial edema also led to decreased heart rate and decreased erythrocyte flow. These compounds which are also considered to be environmental pollutants which includes TCDD (Belair et al., 2001;Prasch et al., 2003;Antkiewicz et al., 2005;Carney et al., 2006), dichlorvos (Sisman, 2010) and BPA (Duan et al., 2008). Similarly, in our studies, defects like heart rate of the zebrafish larvae decreased and blood cell accumulation increased with increase in pericardial edema. ...
MethylParaben (MP), a methyl ester of p-hydroxybenzoic acid, is used as an anti-microbial preservative in foods, drugs and cosmetics for decades. It enters the aquatic environment, and can have toxic effects on aquatic organisms. Little is known on the developmental toxicity of MP exposure to zebrafish during early life stages. In this study, the developmental effects of MP were evaluated in embryo-larval zebrafish (at concentrations ranging from 100 μM, 200 μM, 400 µM, 800 μM and 1000 μM for 96 h post fertilization (hpf). The survival, hatching, heart beat rate and developmental abnormalities were observed in the embryos exposed to MP. MP exposure resulted in decreased heart rate and hatching rate. Defects including pericardial edema blood cell accumulation and bent spine were observed in all the treated concentration, except at 100 μM. With increasing concentrations, the frequency of these defects increased. The 96 hpf LC50 of MP was calculated to be 428 μM (0.065 mg/L). Furthermore, RT-PCR result showed that in larval zebrafish exposed to 100 μM (0.015 mg/L) of MP till 96 hpf, expression of vitellogenin I (Vtg -I) was significantly upregulated compared to the control group. This data suggest that even though lower concentrations of MP do not cause phenotypic malformations, it leads to dysregulated expression of estrogenic biomarker gene Vtg-I.
... The toxic effect of the organic pollutant, dioxin, on human health is well known [58]. The effect of one of the most commonly occurring dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was investigated in zebrafish [59][60][61]. In addition to other developmental defects, zebrafish embryos exposed to TCDD displayed malformed heart, defective atrioventricular valves, and pericardial edema [62]. ...
Environmental pollution is a serious problem of the modern world that possesses a major threat to public health. Exposure to environmental pollutants during embryonic development is particularly risky. Although many pollutants have been verified as potential toxicants, there are new chemicals in the environment that need assessment. Heart development is an extremely sensitive process, which can be affected by environmentally toxic molecule exposure during embryonic development. Congenital heart defects are the most common life-threatening global health problems, and the etiology is mostly unknown. The zebrafish has emerged as an invaluable model to examine substance toxicity on vertebrate development, particularly on cardiac development. The zebrafish offers numerous advantages for toxicology research not found in other model systems. Many laboratories have used the zebrafish to study the effects of widespread chemicals in the environment on heart development, including pesticides, nanoparticles, and various organic pollutants. Here, we review the uses of the zebrafish in examining effects of exposure to external molecules during embryonic development in causing cardiac defects, including chemicals ubiquitous in the environment and illicit drugs. Known or potential mechanisms of toxicity and how zebrafish research can be used to provide mechanistic understanding of cardiac defects are discussed.
... Due to the similarity of its genome structure with humans and other vertebrates, and almost the same metabolism and embryonic development, the zebrafish became a test organism utilized frequently in toxicology studies (Kimmel et al., 1995;Lele and Krone, 1996;Belair et al., 2001;Andreasen et al., 2002;Stern and Zon, 2003;Bello et al., 2004;Balasubramani and Pandian, 2008;Lawson et al., 2011;Chang et al., 2013;Lu et al., 2014). ...
In this study, histopathological changes induced by lindane a gamma isomer of hexachlorocyclohexane and its intoxication associated with reproductive system and hormone levels were investigated in zebrafish. In all, 320 zebrafish adults approximately 1 year in age, (Danio rerio) obtained from a commercial entity were used in the study. Zebrafish were divided into 8 groups, each one containing 40 zebrafish. Groups were organized as 0 (the control group), 1 ml/L methanol, and 5, 10, 20, 40, 80, 160 μg/L/day lindane. In the study, the gamma-HCH-isomer was applied at rates of 5/10/20/40/80/160 μg/L/day as doses of immersion to each group for 21 days. Macroscopically, the drop in egg production was observed, especially in the 80 and 160 μg/L/day groups of female zebrafish. A microscopic decrease in ovulation and biochemical decreases in estradiol (E2) levels (40/80/160 μg/L) were also observed in the female zebrafish. As a result, lindane was found to cause changes in the reproductive system, and consequently, to cause hormonal disorders and to have significant effects on ovulation and fertilization in the female zebrafish. © Published by Central Fisheries Research Institute (CFRI) Trabzon, Turkey.
... B(a)p generally caused oxidative damage to the organisms through biological transformation processes such as lipid peroxidation or DNA damage (Mitchelmore et al. 1998). Py had an effect on the development-related enzyme CYPIA via AhR, resulting in multiple malformations at the early developmental stages, such as peripheral circulatory disorders, anaemia or abnormal cell death due to pericardial sac oedema and neural tube defects (Belair et al. 2001). Phe, with its small molecular weight, produced toxic effects on aquatic animals For example, zebrafish embryos and larvae exposed to Phe can develop malformations causing problems such as cardiac dysfunctions. ...
The red sea bream (Pagrosomus major) 48-h embryo-larval bioassay was used to assess and compare the developmental toxicities of eight typical organic pollutants, including polycyclic aromatic hydrocarbons (PAHs), organophosphorus pesticides, polychlorinated biphenyls (PCBs) and alkylphenol. Toxicological endpoints such as survival rates of P. major embryos or larvae and the rates of hatching and of malformation (oedema, condensed blood, spinal curvatures or eye abnormalities) were noted and described within 48 h of exposure. The LC50, EC50, no-observed-effect concentration (NOEC) and lowest-observed-effect concentration (LOEC) were calculated, based on the dose-response relationship. The results showed that exposures to all of the selected organic pollutants except for methyl parathion produced acute toxic effects on P. major embryos and larvae, at different levels of exposure. The levels of acute toxicity of the eight typical organic pollutants for P. major embryos and larvae showed the following trend: benzo(a)pyrene > malathion > PCB 126 > pyrene > nonylphenol > phenanthrene > monocrotophos > methyl parathion. However, the larvae were more sensitive to these pollutants than embryos, according to the calculated LC50, EC50, NOEC and LOEC. This increased sensitivity of larvae could have resulted from losing the natural barrier function of the egg shell membrane. Benzo(a)pyrene, malathion, nonylphenol and monocrotophos delayed the development of P. major for both embryos and larvae, and decreased the hatching rate of the embryos. These results implied that the development of fish embryos and larvae could serve as potential biomarkers for evaluating organic contamination in the aquatic environment. The marine economic fish P. major was more sensitive to PAHs than the model fish, marine medaka (Oryzias melastigma). The estimated safe concentrations (SCs) for marine economic fish, as determined in our research, could provide a reference for the formulation of water quality criteria.
... In our study, a steep increase in the sub-lethal effects, such as blood flow, heart oedema, malformed yolk, and bent spine was observed after 72 h compared to 48 h exposure. This finding is comparable to the studies of Antkiewicz et al. (2005) and Belair et al. (2001), where TCDD exposure reduced the blood flow in zebrafish embryos after 72 h post-fertilization. Similar findings were observed in the study of Schrankel et al. (1982), where chick embryo lethality occurred within three to four days after treatment with TCAB, and more embryos died until the twelfth day of incubation. ...
... However, Winata et al. (2010) showed that normal blood circulation plays an important role in swim bladder development. This is significant because TCDD causes heart malformations that culminate in heart failure and a complete loss of circulation (Antkiewicz et al., 2005;Belair et al., 2001;Henry et al., 1997). Therefore, we hypothesized that TCDDinduced heart failure impairs development of the swim bladder secondary to circulatory failure. ...
The swim bladder is a gas-filled organ that is used for regulating buoyancy and is essential for survival in most teleost species. In zebrafish, swim bladder development begins during embryogenesis and inflation occurs within 5 days post fertilization (dpf). Embryos exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) before 96h post fertilization (hpf) developed swim bladders normally until the growth/elongation phase, at which point growth was arrested. It is known that TCDD exposure causes heart malformations that lead to heart failure in zebrafish larvae, and that blood circulation is a key factor in normal development of the swim bladder. The adverse effects of TCDD exposure on the heart occur during the same period of time that swim bladder development and growth occurs. Based on this coincident timing, and the dependence of swim bladder development on proper circulatory development, we hypothesized that the adverse effects of TCDD on swim bladder development were secondary to heart failure. We compared swim bladder development in TCDD-exposed embryos to: (1) silent heart morphants, which lack cardiac contractility, and (2) transiently transgenic cmlc2:caAHR-2AtRFP embryos, which mimic TCDD-induced heart failure via heart-specific, constitutive activation of AHR signaling. Both of these treatment groups, which were not exposed to TCDD, developed hypoplastic swim bladders of comparable size and morphology to those found in TCDD-exposed embryos. Furthermore, in all treatment groups swim bladder development was arrested during the growth/elongation phase. Together, these findings support a potential role for heart failure in the inhibition of swim bladder development caused by TCDD.
Copyright © 2015 Elsevier B.V. All rights reserved.
... Using zebrafish as a model, it was quickly determined that TCDD exposure during early development causes decreased cardiomyocyte proliferation, a block and lack of erythrocyte development, reduced blood flow and cardiac output, and lack endocardial valve cushions. Ultimately, this leads to ventricular standstill and death [43,[51][52][53][54][55]. Understanding why TCDD and related AHR agonists cause cardiotoxicity has been a question for quite some time. ...
Zebrafish (Danio rerio) are an excellent vertebrate model for studying heart development, regeneration and cardiotoxicity. Zebrafish embryos exposed during the temporal window of epicardium development to the aryl hydrocarbon receptor (AHR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exhibit severe heart malformations. TCDD exposure prevents both proepicardial organ (PE) and epicardium development. Exposure later in development, after the epicardium has formed, does not produce cardiac toxicity. It is not until the adult zebrafish heart is stimulated to regenerate does TCDD again cause detrimental effects. TCDD exposure prior to ventricular resection prevents cardiac regeneration. It is likely that TCDD-induced inhibition of epicardium development and cardiac regeneration occur via a common mechanism. Here, we describe experiments that focus on the epicardium as a target and sensor of zebrafish heart toxicity.
... Piscine, avian, and mammalian cardiovascular systems are sensitive to TCDD toxicity, with effects including cardiac enlargement, edema, and several dysfunctions. In zebrafish embryos, these effects include a reduction in cardiomyocyte number at 48 hpf, decreased heart size, altered vascular remodeling, pericardial edema, and decreased ventricular contraction culminating in ventricular standstill [31][32][33][34]. ...
Zebrafish is developing as a major model for assessing toxicity of pharmaceuticals, drugs, and pollutants. Besides its applications in regulatory toxic-ity and drug discovery, its characteristics make it a unique system to analyze sublethal toxic effects that only can be studied applying holistic, in toto approaches. Here, we show some of these analyses, in which complex organic systems (neuro-nal, muscular, sensorial, digestive, thyroid), as well as the embryonic development, show specific effects upon exposure to pharmaceuticals and several environmen-tally relevant substances, including nanoparticles and other emerging pollutants for which no adequate toxicological profile is still available. These analyses are especially relevant for embryo risk evaluation, given the close similarity of the early stages of the development in all vertebrates, including humans.
... a p ≤ 0.05 TCDD versus DMSO; *p ≤ 0.05 AHRRa-MO group versus no-MO treatment. CYP1 expression data from these experiments was reported previously ruption of definitive erythropoiesis has been observed in zebrafish embryos in which AHR has been activated exogenously, by exposure to the AHR agonist TCDD (Belair et al., 2001). Similarly, chronic TCDD exposure caused down-regulation of adult hemoglobins in juvenile rainbow trout (Liu et al., 2013). ...
The aryl hydrocarbon receptor repressor (AHRR) is a transcriptional repressor of aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor (HIF) and is regulated by an AHR-dependent mechanism. Zebrafish (Danio rerio) possess two AHRR paralogs; AHRRa regulates constitutive AHR signaling during development, while AHRRb regulates polyaromatic hydrocarbon-induced gene expression. However, little is known about the endogenous roles and targets of AHRRs. The objective of this study was to elucidate the role of AHRRs during zebrafish development using a loss-of-function approach followed by gene expression analysis. Zebrafish embryos were microinjected with morpholino oligonucleotides (MO) against AHRRa or AHRRb to knock down AHRR protein expression. At 72 hours post-fertilization (hpf), microarray analysis revealed that the expression of 279 and 116 genes was altered by knockdown of AHRRa and AHRRb, respectively. In AHRRa-morphant embryos, 97 genes were up-regulated and 182 genes were down-regulated. Among the down-regulated genes were several related to photoreceptor function, including cone-specific genes such as several opsins (opn1sw1, opn1sw2, opn1mw1, opn1lw2), phosphodiesterases (pde6H, pde6C), retinol binding protein (rbp4l), phosducin, and arrestins. Down-regulation was confirmed by RT-PCR and with samples from an independent experiment. The four genes tested (opn1sw1, pde6H, pde6C, arr3b) were not inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin. AHRRa knockdown also caused up-regulation of embryonic hemoglobin (hbbe3), suggesting a role for AHRR in regulating hematopoiesis. Knockdown of AHRRb caused up-regulation of 31 genes and down-regulation of 85 genes, without enrichment for any specific biological process. Overall, these results suggest that AHRRs may have important roles in development, in addition to their roles in regulating xenobiotic signaling.
... This observed reduction of pericardial and sub-epithelial oedema as yolk-sac larvae develop appears to reflect an increasing ability to maintain ionic and osmotic balance throughout the yolk-sac period. In agreement, oedema is not observed in zebrafish larvae (Danio rerio) after exposure to contaminants if exposure is delayed during ontogeny suggesting that larvae are particularly vulnerable shortly after hatching (Belair et al., 2001). ...
The Nile tilapia, one of the most extensively cultured tropical freshwater species, offers considerable potential for culture in low-salinity water. The ontogeny of osmoregulation in the Nile tilapia was studied from spawning to yolk-sac absorption after exposure to different experimental conditions ranging from freshwater to 25 ppt. Eggs were able to withstand elevated rearing salinities up to 20 ppt, but transfer to 25 ppt induced 100% mortality by 48 h post-fertilization. Across all stages, embryos and larvae hyper-regulated at lower salinities and hypo-regulated at higher salinities. Osmoregulatory capacity increased during development and from 2 days post-hatch onwards remained constant until yolk-sac absorption. Adjustments of larval osmolality following abrupt transfer from freshwater to experimental salinities (12.5 and 20 ppt), followed a pattern of crisis and regulation, with values for larvae stabilising at c. 48 h post-transfer for all treatments, regardless of age at time of transfer. Age at transfer to experimental salinities (7.5–20 ppt) had a significant positive effect on larval ability to osmoregulate, with larvae transferred at 8 days post-hatch maintaining more constant whole-body osmolality over the experimental salinities tested than larvae at hatch. Concomitantly, survival following transfer to experimental salinities increased with age. There was a significant effect (GLM; p < 0.05) of the salinity of incubation and rearing on the incidence of gross larval malformation that was seen to decline over the developmental period studied.
... et al., 2010), the extent to which CTN adversely affected cardiovascular function was evaluated by using Tg(gata1:dsRed) line with red fluorescence in erythrocytes. Direct visualization of fluorescence intensity in the axial vessels of zebrafish is an indicator of blood circulation (Belair et al., 2001;Fish et al., 2011). Reduction of fluorescence in CTN-treated 48 hpf embryos suggests that CTN could attenuate the blood flow rate (Fig. 3A). ...
Citrinin (CTN) is a fungal secondary metabolite which contaminates various foodstuffs and animal feeds; it also exhibits organotoxicity in several animal models. In this study, the zebrafish was used to elucidate the mechanism of CTN cardiotoxicity in developing embryos. Following CTN administration, the gross morphology of the embryonic heart was apparently altered, including heart malformation, pericardial edema and red blood accumulation. Whole-mount immunostaining and histological analysis of ventricle and atrium indicated incorrect heart looping and reduced size of heart chambers. From the perspective of cardiac function, the heartbeat and blood flow rate of embryos were significantly decreased in the presence of CTN. CTN also modulated the expression of tbx2a and jun B genes, but not that of bmp4 and nkx2.5. Furthermore, the heart areas of CTN-exposed embryos demonstrated an elevated levels of aldh1a2 and cspg2 mRNA; these two cardiac-related genes are known to be involved in retinoic acid (RA) pathway as well as downstream targets of microRNA-138 (miR-138) in zebrafish. CTN treatment also down regulated the expression of miR-138. Interestingly, over-expression of miR-138 was able to rescue the heart defects generated by CTN. These results support the notion that CTN exposure has a severe impact on heart development, affecting heart morphogenesis through the dysregulation of miR-138, RA signaling and tbx2a.
... We have previously focused on the acute effects of TCDD exposure on zebrafish embryos (Antkiewicz et al., 2005;Belair et al., 2001;Carney et al., 2006a;Xiong et al., 2008). Because it is clear that TCDD exposure alters developmental processes, it seems likely that even sublethal exposure could produce permanent effects. ...
The acute effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure have been well documented in many vertebrate species. However, less is known about the consequences in adulthood from sublethal exposure during development. To address this, we exposed zebrafish to sublethal levels of TCDD (1 h; 50 pg/ml), either in early embryogenesis (day 0) or during sexual determination (3 and 7 weeks), and assessed the effects later in adulthood. We found that exposure during embryogenesis produced few effects on the adults themselves, but did affect the offspring of these fish: malformations and increased mortality were observed in the subsequent generation. Zebrafish exposed during sexual development showed defects as adults in the cranial and axial skeleton. This was most clearly manifested as scoliosis caused by malformation of individual vertebrae. These fish also showed defects in reproduction, producing fewer eggs with lower fertilization success. Both males and females were affected, with males contributing to the decrease in egg release from the females, and exposed females contributing to fertilization failure. TCDD exposure at 3 and 7 weeks produced feminization of the population. Surprisingly, part of this was due to the appearance of fish with clearly female bodies, yet carrying testes in place of ovaries. Our results show that exposures that produce little if any impact during development cause can cause severe consequences during adulthood, and present a model for studying this process.
... Zebrafish exposed to TCDD immediately after fertilization develop edema as early as 72 hpf (Henry et al., 1997). Interestingly, if exposure to TCDD is delayed until after 96 hpf, edema is not observed (Belair et al., 2001). This suggests that developing zebrafish are especially vulnerable to TCDD shortly after hatching. ...
Zebrafish have been used predominantly in developmental biology and molecular genetics, but their value in toxicology as well as drug discovery has been recognized. To evaluate the toxicity of a chemical, it is essential to identify the endpoints of toxicity and their dose-response relationships, elucidate the mechanisms of toxicity, and determine the toxicodynamics of the chemical. In addition to detailed toxicological investigations of a single chemical, there also is a need for high-throughput large- scale screening for toxicity of several hundreds of chemicals at a time. In both cases, the zebrafish has numerous attributes. More is probably known about ''what is normal'' in the zebrafish than any other fish species. This includes morpho- logical, biochemical, and physiological information at all stages of early development and in juveniles and adults of both sexes. This makes using the zebrafish ideal for toxicology research where the objective is to identify adverse effects of chemical exposure.
... These dioxin-like compounds (DLCs) are persistent and widespread pollutants that accumulate in fish tissues due to their lipophilic properties. Responses of early life stages of fish to PHHs include induction of cytochrome P4501A (CYP1A) detoxification enzymes, developmental arrest, craniofacial deformities, pericardial and yolk sac edemas, hemorrhages and mortality (Belair et al., 2001;Henry et al., 1997;Hill et al., 2004a,b;Teraoka et al., 2002). Lethal embryotoxicity of PHHs may have contributed to lake trout (Salvelinus namaycush) decline in their TCDD toxic equivalents concentration (TCDD-TEQs), i.e. the sum of each PHH concentration times its toxic equivalent factor (TEF; relative toxicity of each PHH versus TCDD) (van Zorge et al., 1989). ...
The relative potency (ReP) of 3,3',4,4',5-pentachlorobiphenyl (PCB126) to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for sublethal responses was assessed in Fundulus heteroclitus embryos. Eggs were treated with intravitelline injections of graded sublethal doses of PCB126 (312-5000pgg(-1) wet weight, ww) or TCDD (5-1280pgg(-1) ww). At 16 days post-fertilization (DPF), craniofacial deformities were observed in larvae hatched from eggs treated with the two highest doses of PCB126 (2500-5000pgg(-1) ww). Both compounds caused a dose-responsive reduction of larval growth and prey capture ability (at ≥1250pgg(-1) ww), and induction of ethoxyresorufin-O-deethylase (EROD) activity (at ≥80pgg(-1) ww). The dose-response relationships for EROD activity for PCB126 and TCDD had similar slopes and the ReP of PCB126 to TCDD for EROD activity was estimated at 0.71. This is 140-fold higher than the World Health Organization (WHO) TCDD equivalency factor (TEF) of PCB126 for fish (0.005), which is based on rainbow trout (Oncorhynchus mykiss) embryolethality data. The slope of the dose-response relationship for prey capture ability for PCB126 was steeper than for TCDD, suggesting different mechanisms of action. Expression levels of several genes were also studied by quantitative real-time polymerase chain reaction (qPCR) following exposure to single doses of TCDD or PCB126 (1280 and 1250pgg(-1) ww, respectively) causing similar EROD induction. A different pattern of responses was observed between PCB126 and TCDD: PCB126 appeared to induce antioxidant responses by inducing sod2 expression, while TCDD did not. These results suggest that relative potencies are species-specific and that the current ReP for PCB126 underestimates its toxicity for some fish species. It is recommended to develop species-specific RePs for a variety of sublethal endpoints and at environmentally relevant doses.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant that activates the aryl hydrocarbon receptor (AhR) pathway, has been reported to cause cardiac damage. However, the mechanism underlying AhR-induced cardiac defects in response to TCDD exposure remains unclear. In this study, we characterized the impacts of TCDD exposure on heart morphology and cardiac function in zebrafish. TCDD exposure in the early developmental stage of zebrafish embryos led to morphological heart malformation and pericardial edema, concomitant with reduced cardiac function. These cardiac defects were attenuated by inhibiting AhR activity with CH223191. Transcriptome profiling showed that, along with an upregulation of the AhR signaling pathway by TCDD treatment, the expression of pro-ferroptotic genes was upregulated, while that of genes implicated in glutathione metabolism were downregulated. Moreover, lipid peroxidation, as indicated by malonaldehyde (MDA) production, was increased in TCDD-exposed cardiac tissue. Accordingly, inhibiting lipid peroxidation with liproxstatin-1 reversed the adverse cardiac effects induced by TCDD treatment. Taken together, our findings demonstrate that AhR-mediated lipid peroxidation contributes to cardiac defects in the early developmental stage in zebrafish embryos exposed to TCDD.
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants that can enter aquatic environments through runoff, atmospheric deposition, accidental discharge, and oil spills. These compounds can be oxidized photochemically or biologically into oxygenated PAHs (oxy-PAHs) which have been shown to be more toxic compared to parent PAHs. The polar properties of oxy-PAHs increase their mobility within the environment which increases the risk of exposure to fauna and flora compared to parent PAHs. Regioselective toxicity has been observed in several oxy-PAHs and the oxidation state of the oxygen on a specific PAH can have dramatic impacts on the toxicity. Previous studies have found that exposure to hydroxychrysenes at critical developmental time-points in fish models impairs red blood cell concentrations in a regioselective manner, with 2-hydroxychrysene (2-OHCHR) being more potent than 6-hydroxychrysene (6-OHCHR). The mechanisms of toxicity of oxy-PAHs are largely unknown and we aimed to characterize the pathways of toxicity of 2- and 6-OHCHR in fish embryos. Our first aim was to characterize the toxic effects in Japanese medaka embryos and to find a sensitive window of development to hydroxychrysenes. We found that 2-OHCHR caused anemia and morality in medaka embryos in contrast to in zebrafish embryos, where 2-OHCHR caused only anemia and 6-OHCHR only caused mortality. A sensitive window to 2-OHCHR toxicity was found between 52-100 hpf which closely coincided with liver development. This led us to our second aim, exploring the metabolism and toxicokinetics of the hydroxychrysenes. We found that although 6-OHCHR was taken up 97.2% ± 0.18 more rapidly than 2-OHCHR, it was also eliminated 57.7% ± 0.36 faster as a glucuronide conjugate. Pretreatment with the general cytochrome P450 inhibitor ketoconazole reduced anemia by 96.8% ± 3.19 and mortality by 95.2% ± 4.76 of 2-OHCHR treatments. In addition, formation of the 1,2-catechol was also reduced by 64.4% ± 2.14. However, while pretreatment with the UGT inhibitor nilotinib reduced glucuronidation of 2-OHCHR by 52.4% ± 2.55 and of 6-OHCHR by 63.7% ± 3.19, it did not alter toxicity for either compound. These results indicated that CYP mediated activation, potentially to the oxidatively active metabolite 1,2-catechol, may be driving the isomeric differences in toxicity. Previous studies have found 2-OHCHR to be a four-fold more potent aryl hydrocarbon receptor (AhR) agonist compared to 6-OHCHR. Therefore, in aim 3, we explored the role of the and oxidative stress in 2-OHCHR toxicity. While treatments with the AhR agonists PCB126 and 2-methoxychrysene (2-MeOCHR) did not cause significant anemia or mortality, pretreatments with AhR antagonist CH-223191 reduced anemia by 97.2% ± 0.84 and mortality by 96.6% ± 0.69. AhR inhibition was confirmed by a significant reduction (91.0% ± 9.94) in EROD activity. Thiobarbituric acid reactive substances (TBARS) concentrations were 32.9% ± 3.56 higher (p<0.05) in 2-OHCHR treatments at 100 hpf compared to controls, indicating oxidative stress. Staining with 2’,7’-Dichlorofluorescin diacetate (DCFDA) revealed 42.6% ± 2.69 of embryos exhibiting high concentrations of ROS in caudal tissues, which is a site for embryonic hematopoiesis. Both muscle and skeletal tissues were affected, as well as some caudal vasculature. Overall, our findings indicate that AhR may mediate 2-OHCHR toxicity, upregulating CYP and potentially forming the 1,2-catechol that generates ROS in the embryos within caudal tissues, potentially disrupting hematopoiesis leading to anemia and subsequent mortality. Further studies should investigate additional key events and construct adverse outcome pathways for oxy-PAHs.
The aryl hydrocarbon receptor (AHR) plays an important role during mammalian embryo development. Inhibition of AHR signaling promotes the development of hematopoietic stem/progenitor cells. AHR also regulates the functional maturation of blood cells, such as T cells and megakaryocytes. However, little is known about the role of AHR modulation during the development of erythroid cells. In this study, we used the AHR antagonist StemRegenin 1 (SR1) and the AHR agonist 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) during different stages of human erythropoiesis to elucidate the function of AHR. We found that antagonizing AHR signaling improved the production of human embryonic stem cell (hESC)-derived erythrocytes and enhanced erythroid terminal differentiation. RNA-sequencing showed that SR1 treatment of proerythroblasts upregulated the expression of erythrocyte differentiation-related genes and downregulated actin organization-associated genes. We found that SR1 promoted F-actin remodeling in terminally differentiated erythrocytes, favoring the maturation of the cytoskeleton and enucleation. We demonstrated that the effects of AHR inhibition on erythroid maturation resulted from an increase in F-actin remodeling. Our findings help uncover the mechanism for AHR-mediated human erythroid cell differentiation. We also provide a new approach toward the large-scale production of functionally mature hPSC-derived erythrocytes for use in translational applications.
The fish embryo acute toxicity (FET) test is known to be less sensitive than the fish acute test for some chemicals, including neurotoxicants. Thus, there is an interest in identifying additional endpoints that can improve FET test performance. The goal of this project was to advance alternative toxicity testing methods by determining whether select developmental abnormalities-snout-vent length, eye size, and pericardial area-are linked to adverse alterations in ecologically-relevant behaviors and delayed mortality. Fathead minnow (Pimephales promelas) FET tests were conducted with 3,4-dicholoroaniline, cadmium, and perfluorooctanesulfonic acid (PFOS) and developmental abnormalities were quantified. Surviving eleutheroembryos were reared in clean water to 14 days post fertilization (dpf), during which time behaviors and mortality were evaluated. None of the abnormalities evaluated were predictive of behavioral alterations; however, embryos with ≥14% reductions in length or ≥3.54-fold increases in pericardial area had an 80% chance of mortality by 14 dpf. When these abnormalities were used as markers of mortality, the LC50s for cadmium and PFOS were less than those calculated using only standardized FET test endpoints and similar to those obtained via larval fish tests, indicating that the snout-vent length and pericardial area warrant consideration as standard FET test endpoints.
The aryl hydrocarbon receptor (AHR) has endogenous functions in mammalian vascular development and is necessary for mediating the toxic effects of a number of environmental contaminants. Studies in mice have demonstrated that AHR is necessary for the formation of the renal, retinal, and hepatic vasculature. In fish, exposure to the prototypic AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces expression of the AHR biomarker cyp1a throughout the developing vasculature and produces vascular malformations in the head and heart. However, it is not known whether the vascular structures that are sensitive to loss of AHR function are also disrupted by aberrant AHR activation. Here, we report that TCDD-exposure in zebrafish disrupts development of 1) the subintestinal venous plexus (SIVP), which vascularizes the developing liver, kidney, gut, and pancreas, and 2) the superficial annular vessel (SAV), an essential component of the retinal vasculature. Furthermore, we determined that TCDD exposure increased the expression of bmp4, a key molecular mediator of SIVP morphogenesis. We hypothesize that the observed SIVP phenotypes contribute to one of the hallmarks of TCDD exposure in fish – the failure of the yolk sac to absorb. Together, our data describe novel TCDD-induced vascular phenotypes and provide molecular insight into critical factors producing the observed vascular malformations.
Highlighting latest advances in genetics and biochemistry, the completely revised Third Edition reviews the field from basic science, clinical, epidemiological, and regulatory perspectives. Contributions from top opinion leaders in the field bring together developments in molecular embryology and cell biology as they apply to problems in developmental toxicology. It covers testing of pharmaceutical and environmental agents and interpretation of developmental toxicology data, highlighting mathematical and statistical techniques, as well as the effects of toxic exposure on the functional development of various organs. The relationship between maternal and developmental toxicology is examined, in addition to current techniques for studying chemical disposition, metabolism, and placental transfer. Close attention is given to the regulatory aspects of testing and risk assessment. Pre and postconceptional clinical care and genetic factors in clinical developmental toxicology are also discussed.
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that binds environmental toxins and regulates gene expression. AHR also regulates developmental processes, like craniofacial development and hematopoiesis, in the absence of environmental exposures. Zebrafish have three paralogues of AHR: ahr1a, ahr1b and ahr2. Adult zebrafish with mutations in ahr2 exhibited craniofacial and fin defects. However, the degree to which ahr1a and ahr1b influence ahr2 signaling and contribute to fin and craniofacial development are not known. We compared morphology of adult ahr2 mutants and ahr1a/ahr1b single and double mutant zebrafish. We found that ahr1a/ahr1b single and double mutants were morphologically normal while ahr2 mutant zebrafish demonstrated fin and craniofacial malformations. At 5 days post fertilization, both ahr1a/ahr1b and ahr2 mutant larvae were normal, suggesting that adult phenotypes are due to defects in maturation or maintenance. AHR was shown to interact with estrogen receptor alpha, yet it is not known whether these interactions are constitutive or dependent on ahr1 genes. To determine whether estrogen receptors are constitutive cofactors for AHR signaling, we used genetic and pharmacologic techniques to analyze TCDD-dependent toxicity in estrogen receptor and ahr mutant embryos. We found that embryos with mutations in ahr1a/ahr1b or estrogen receptor genes are susceptible to TCDD toxicity while ahr2 mutant embryos are TCDD-resistant. Moreover, pharmacologic blockade of nuclear estrogen receptors failed to prevent TCDD toxicity. These findings suggest that ahr1 genes do not have overlapping functions with ahr2 in fin and craniofacial development or TCDD-dependent toxicity, and that estrogen receptors are not constitutive partners of ahr2.
Due to the ever-increasing number of chemicals coming to market, and the cost of performing traditional in vivo studies, there has been a shift toward the use of less costly alternative techniques. The adverse outcome pathway (AOP) concept has emerged as a scaffold for organizing mechanistic information from these methods. Two main elements – key events (KEs) and key event relationships (KERs) – are utilized to describe the underlying mechanism outlined by the AOP. Each KE depicts the measureable changes in the state of the biological system at each level of organization that are essential for the progression along the pathway. The KERs, meanwhile, contain the biological information that connects each of the KEs. This chapter covers some of the potential applications for AOPs when performing risk assessment of chemical mixtures. The structure of the AOP provides much more precision when considering mechanistic data in a mixtures assessment. The use of this concept provides a means to allow more specificity when deciding whether to use dose addition, independent action or integrated addition risk assessment methodologies. Furthermore, AOPs enable novel approaches for determining chemical groups and how they may be utilized within mixtures risk assessment.
Angiogenesis is one of the major mechanisms of neovascularization to form newly sprouting capillaries from the originally existing microvessels, which covers a broad spectrum of physiological and pathophysiological processes from tissue hypertrophy and wound healing to inflammation and even cancer. A recent report estimates that a quarter of the global burden of disease was due to environmental risk factors, and environmental exposures are increasingly recognized to be strongly associated with major human systemic diseases, such as cardiovascular disorders and cancers, especially in susceptible populations. Therefore, angiogenesis and its regulatory factors, along with major environmental exposure factors, such as cigarette smoke, ambient air pollutants, and some major carcinogens, are reviewed.
Dioxin exposure and its effect on hematopoiesis and cancer have been largely investigated in both human and non-human settings. Here we systematically reviewed literature to address the question of what we know about TCDD biology and exposure. Most effects are due to TCDD interaction with a receptor of xenobiotics called AHR, which is ubiquitously represented and also works on hematopoietic myeloid and lymphoid stem cells, inducing proliferation and stem cell release from bone marrow to peripheral circulation. Epidemiologic studies on TCDD exposure demonstrated an association with onco-hematologic diseases, particularly with non Hodgkin lymphomas and multiple myeloma, and non hematologic cancers, such as sarcomas, although these relationships are affected by multiple confounding factors.
The molecular program controlling hematopoietic differentiation is not fully understood. Here, we describe a family of zebrafish genes that includes a novel hematopoietic regulator, draculin-like 3 (drl.3). We found that drl.3 is expressed in mesoderm-derived hematopoietic cells and is retained during erythroid maturation. Moreover, drl.3 expression correlated with erythroid development in gata1a- and spi1b-depleted embryos. Loss-of-function analysis indicated that drl.3 plays an essential role in primitive erythropoiesis and, to a lesser extent, myelopoiesis that is independent of effects on vasculature, emergence of primitive and definitive progenitor cells and cell viability. While drl.3 depletion reduced gata1a expression and inhibited erythroid development, enforced expression of gata1a was not sufficient to rescue erythropoiesis, indicating that the regulation of hematopoiesis by drl.3 extends beyond control of gata1a expression. Knockdown of drl.3 increased the proportion of less differentiated, primitive hematopoietic cells without affecting proliferation, establishing drl.3 as an important regulator of primitive hematopoietic cell differentiation.
The documented 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD)‐induced effects on zebrafish Danio rerio including craniofacial malformations and a general retardation of growth, were further characterized in the present study. A significant decrease in total body length and the length of each bone in the upper and lower jaw was identified in exposed larvae from an exposure concentration of 30 ng l−1 TCDD. This study is the first quantitative evidence for the effects of TCDD on the upper jaw and also demonstrates that TCDD‐induced craniofacial malformations and retardation of growth are very sensitive endpoints of dioxin toxicity.
A transgenic zebrafish strain was generated expressing the aryl hydrocarbon receptor (AhR)-regulated green fluorescent protein (GFP) reporter gene. Following exposure to β-naphthoflavone (βNF), the transgenic fish exhibited up-regulation of GFP in the face and vertebrae compared to vehicle controls. βNF-exposed fish exhibited gross dysmorphogenesis in vertebral development by 5 days after fertilization.
In the present study, we have used salmon embryos whose continuous exposure to waterborne PFOA or PFOS at 100μg/L started as freshly fertilized eggs, and lasted for a total of 52 days. PFOS and PFOA were dissolved in methanol (carrier vehicle) whose concentration never exceeded 0.01% of total tank volume. Samples were collected at day 21, 28, 35, 52, 49 and 56 after the start of the exposure. Note that days 49 and 56 represent end of exposure and 1 week after a recovery period, respectively. Tissue bioaccumulations were determined by HPLC/MS/MS, steroid hormones, fatty acids (FAs) and lipids were determined by GC-MS, while mRNA expression levels of genes were determined by qPCR in whole body homogenate. We observed that PFOS and PFOA showed a steady increase in whole body burden during the exposure period, with a slight decrease after the recovery period. Calculated somatic indexes showed that PFOA produced increases in heart-, thymus-, liver- and kidney somatic indexes (HSI, TSI, LSI and KSI). PFOA and PFOS exposure produced significant decreases in whole body dehydroepiandrosterone (DHEA), estrone and testosterone at sampling day 21 and a strong increase of cortisol and cholesterol at the end of recovery period (day 56). PFOA and PFOS effects differed with DHEA and estrone. While PFOS decreased DHEA levels, PFOA produced an increase at day 49, and while PFOS decreased estrone, PFOA produced a slight increase at day 56. We observed changes in FA composition that predominantly involved increases in FA methyl esters (FAMEs), mono- and poly-unsaturated FA (MUFA and PUFA) and a decrease in n-3/n-6 PUFA ratio by both PFOA and PFOS. Particularly, an increase in - pentadecenoic MUFA (15:1), two n-3 PUFAs α-linolenic acid [ALA: 18:3 n3] and eicosapentaenoic acid [EPA: 20:5 n-3] and n-6 PUFA: arachidonic acid [ARA: 20:4 n6], docosapentaenoic acid (DPA) by PFOA and PFOS were observed. These effects were associated with changes in mRNA expression of FA elongase (FAE), Δ5-desaturase (FAD5) and Δ6-desaturase (FAD6) genes. In summary, the changes in hormonal and FA profiles may represent cellular and/or physiological adaptation to continuous PFOS and PFOA exposure by increasing membrane fluidity, and/or overt developmental effects. The present findings provide some potential insights and basis for a better understanding on the possible mechanisms of PFCs toxicity in fish.
Lake trout (Salvelinus namaycush) eggs containing [3H]TCDD concentrations from 0 to 302 parts per trillion (ppt) were observed through the fry stage for TCDD metabolism, elimination, and toxicity. All radioactive residues extracted from eggs and sac fry were due to TCDD; no metabolites were detected. [3H]TCDD was not eliminated from eggs and sac fry, but was rapidly eliminated from fry (t1/2, 35–37 d). Hatchability was less at egg TCDD concentrations ; however, the greatest TCDD-related mortality occurred during the sac fry stage. In all TCDD groups (34–302 ppt), sac fry that died developed subcutaneous yolk sac edema prior to death, resembling blue-sac disease. The development of yolk sac edema preceded sac fry mortality, and the severity of edema varied directly with cumulative mortality. Based on TCDD concentrations in the egg resulting from a 48-h exposure, the no observable adverse effect level (NOAEL) for mortality was 34 ppt and the lowest observable adverse effect level (LOAEL) was 55 ppt. The TCDD concentration in eggs that caused 50% mortality above control at swim-up (LD50) was 65 ppt. Lake trout sac fry exposed as eggs are more sensitive to the lethal effects of TCDD than any mammalian, avian, or fish species investigated thus far.
Exquisite embryonic lethal mutations have been isolated in hundreds of genes necessary for zebrafish development. Analysis of this resource promises to enhance our understanding of the molecular genetic mechanisms of vertebrate development. This review discusses the state of the zebrafish genome project and the genetic trickery that can expedite molecular isolation of genes disrupted by these mutations.
SCL/Tal-1 is a transcription factor necessary for hematopoietic stem cell differentiation. Although SCL is also expressed in endothelial and neural progenitors, SCL function in these cells remains unknown. In the zebrafish mutant cloche (clo), SCL expression is nearly abolished in hematopoietic and vascular tissues. Correspondingly, it was shown previously that clo fails to differentiate blood and angioblasts. Genetic analysis demonstrates that the clo mutation is not linked to the SCL locus. Forced expression of SCL in clo embryos rescues the blood and vascular defects, suggesting that SCL acts downstream of clo to specify hematopoietic and vascular differentiation.
Developmental toxicity to TCDD-like congeners in fish, birds, and mammals, and reproductive toxicity in mammals are reviewed. In fish and bird species, the developmental lesions observed are species dependent, but any given species responds similarly to different TCDD-like congeners. Developmental toxicity in fish resembles "blue sac disease," whereas structural malformations can occur in at least one bird species. In mammals, developmental toxicity includes decreased growth, structural malformations, functional alterations, and prenatal mortality. At relatively low exposure levels, structural malformations are not common in mammalian species. In contrast, functional alterations are the most sensitive signs of developmental toxicity. These include effects on the male reproductive system and male reproductive behavior in rats, and neurobehavioral effects in monkeys. Human infants exposed during the Yusho and Yu-Cheng episodes, and monkeys and mice exposed perinatally to TCDD developed an ectodermal dysplasia syndrome that includes toxicity to the skin and teeth. Toxicity to the central nervous system in monkey and human infants is a potential part of the ectodermal dysplasia syndrome. Decreases in spermatogenesis and the ability to conceive and carry a pregnancy to term are the most sensitive signs of reproductive toxicity in male and female mammals, respectively.
The aryl hydrocarbon (Ah) receptor has occupied the attention of toxicologists for over two decades. Interest arose from the early observation that this soluble protein played key roles in the adaptive metabolic response to polycyclic aromatic hydrocarbons and in the toxic mechanism of halogenated dioxins and dibenzofurans. More recent investigations have provided a fairly clear picture of the primary adaptive signaling pathway, from agonist binding to the transcriptional activation of genes involved in the metabolism of xenobiotics. Structure-activity studies have provided an understanding of the pharmacology of this receptor; recombinant DNA approaches have identified the enhancer sequences through which this factor regulates gene expression; and functional analysis of cloned cDNAs has allowed the characterization of the major signaling components in this pathway. Our objective is to review the Ah receptor's role in regulation of xenobiotic metabolism and use this model as a framework for understanding the less well-characterized mechanism of dioxin toxicity. In addition, it is hoped that this information can serve as a model for future efforts to understand an emerging superfamily of related signaling pathways that control biological responses to an array of environmental stimuli.
Toxicity and histopathology of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in zebrafish (Danio rerio) early life stages was characterized from 12 to 240 hr postfertilization (hpf) following water-borne exposure of newly fertilized eggs. TCDD did not increase egg mortality (0-48 hpf), nor did it affect time to hatching (48-96 hpf). Egg doses of 1.5 ng [3H]TCDD/g or greater elicited toxic responses in zebrafish larvae. Pericardial edema and craniofacial malformations were first observed at 72 hpf, followed by the onset of yolk sac edema (96 hpf) and mortality (132 hpf). At 240 hpf the ED50s for pericardial edema, yolk sac edema, and craniofacial malformations were 2.2, 2.1, and 1.9 ng [3H]TCDD/g egg, respectively. The LD50, determined at 240 hpf, was 2.5 ng [3H]TCDD/g egg. Severe hemodynamic changes, observed as slowed blood flow in vascular beds of the trunk, head, and gills and slowed heart rate, occurred in TCDD-treated zebrafish prior to or coincident with the onset of gross signs of toxicity. Histological examination of TCDD-treated zebrafish revealed a variety of epithelial tissue lesions including arrested gill development and ballooning degeneration and/or necrosis of the renal tubules, hepatocytes, pancreas, and all major brain regions. Mesenchymal tissue lesions included subcutaneous edema in the head, trunk, and yolk sac, edema of the pericardium and skeletal muscle, and underdevelopment of the swim bladder. This demonstration of zebrafish responsiveness to TCDD early life stage toxicity coupled with the considerable information on developmental biology and genetics of zebrafish provides a foundation for future investigations into the mechanism of TCDD developmental toxicity.
In this study, DNA constructs containing the putative zebrafish promoter sequences of GATA-1, an erythroid-specific transcription factor, and the green fluorescent protein reporter gene, were microinjected into single-cell zebrafish embryos. Erythroid-specific activity of the GATA-1 promoter was observed in living embryos during early development. Fluorescent circulating blood cells were detected in microinjected embryos 24 hours after fertilization and were still present in 2-month-old fish. Germline transgenic fish obtained from the injected founders continued to express green fluorescent protein in erythroid cells in the F1 and F2 generations. The green fluorescent protein expression patterns in transgenic fish were consistent with the pattern of GATA-1 mRNA expression detected by RNA in situ hybridization. These transgenic fish have allowed us to isolate, by fluorescence-activated cell sorting, the earliest erythroid progenitor cells from developing embryos for in vitro studies. By generating transgenic fish using constructs containing other zebrafish promoters and green fluorescent protein reporter gene, it should be possible to visualize the origin and migration of any lineage-specific progenitor cells in a living embryo.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related congeners affect the immune system, causing immunosuppression and thymic atrophy in a variety of animal species. TCDD is believed to exert its effects primarily through the ligand-activated transcription factor, the aryl hydrocarbon receptor (AhR). Although the AhR is found at high levels in both thymocytes and thymic stroma, it is uncertain in which cells TCDD is activating the AhR to cause alterations in the thymus. Some investigators have suggested that stromal elements, primarily epithelial cells, within the thymus are the primary targets for TCDD. Others have suggested that atrophy is due to a direct effect on thymocytes, either by apoptosis or by altering the development of progenitor cells. By producing chimeric mice with TCDD-responsive (AhR[+/+]) stromal components and TCDD-unresponsive (AhR[-/-]) hemopoietic components, or the reverse, we have clarified the role of stromal vs hemopoietic elements in TCDD-induced thymic alterations. Our results show that the targets for TCDD-induced thymic atrophy and phenotypic alterations are strictly in the hemopoietic compartment and that TCDD activation of epithelial cells in the stroma is not required for thymic alterations. Furthermore, changes observed in the putative stem cell populations of these chimeric mice are also dependent on TCDD activation of the AhR in hemopoietic elements.
The SCL gene encodes a basic helix-loop-helix (bHLH) transcription factor that is essential for the development of all haematopoietic lineages. SCL is also expressed in endothelial cells, but its function is not essential for specification of endothelial progenitors and the role of SCL in endothelial development is obscure. We isolated the zebrafish SCL homologue and show that it was co-expressed in early mesoderm with markers of haematopoietic, endothelial and pronephric progenitors. Ectopic expression of SCL mRNA in zebrafish embryos resulted in overproduction of common haematopoietic and endothelial precursors, perturbation of vasculogenesis and concomitant loss of pronephric duct and somitic tissue. Notochord and neural tube formation were unaffected. These results provide the first evidence that SCL specifies formation of haemangioblasts, the proposed common precursor of blood and endothelial lineages. Our data also underline the striking similarities between the role of SCL in haematopoiesis/vasculogenesis and the function of other bHLH proteins in muscle and neural development.
The Human Genome Project is in full swing, and every day we know more of the sequences that define us (Burris et al. 1998). Some smaller bacterial genomes are already sequenced and so are the yeast (S. cerevisiae) and worm (C. elegans) genomes. Progress in sequencing technologies promises that within the next few years we will know the entire sequence of the 3000 Mb human genome, and shortly afterwards the genome sequence of “the queen” of the genetic animal model systems, the mouse. At that point the work on understanding the biological existence of organisms will only begin. The function of genes in patterning, organogenesis, and physiology will need to be uncovered and the best current tools to accomplish these goals lie in mutational analysis of mouse, zebrafish, worm, fly, rat, and, for plants‐Arabidopsis thaliana .A s the complete sequence of each of the model systems becomes known, comparative analyses of gene function will provide clues towards understanding normal development and physiology as well as monogenic traits and complex diseases. Zebrafish was first discovered as a potentially excellent model system to study embryology owing to its transparent embryos
Hematopoiesis in the vertebrate is characterized by the induction of ventral mesoderm to form hematopoietic stem cells and the eventual differentiation of these progenitors to form the peripheral blood lineages. Several genes have been implicated in the differentiation and development of hematopoietic and vascular progenitor cells, yet our understanding of the discrete steps involved in the induction of these cells from the ventral mesoderm is still incomplete. One method of delineating these processes is based on the use of lower vertebrates. The zebrafish (Danio rerio) is an especially robust vertebrate system for both isolating and characterizing genes involved in these processes. Hematopoietic mutants have been generated with defects in many of the steps of both the primitive and definitive hematopoietic programs. Cloning of the genes that underlie these mutations should yield valuable details of hematopoiesis and may have therapeutic implications for bone marrow transplantation and stem cell gene therapy.
The zebrafish vascular and hematopoietic systems are similar in many ways to those of all other vertebrates, including mammals. The study of genes that play important roles in zebrafish vasculogenesis and hematopoiesis is facilitated by many of the unique attributes that the zebrafish offers as a model system. The transparent nature of zebrafish embryos makes it possible to observe blood cells circulating throughout the vasculature from the onset of circulation until at least the end of the larval stage. Genetic approaches can be used to identify mutations that affect virtually all aspects of zebrafish development, including blood vessel formation and hematopoiesis. Screens for mutations that affect these processes are further enhanced by the fact that zebrafish embryos can survive a number of days without a functional cardiovascular system, thus making it possible to study both the early and late effects of vascular and hematopoietic mutations. This characteristic is an important advantage not offered by, for example, the mouse embryo. Combined together, these attributes have propelled the zebrafish forward as a useful model system for the study of these critical processes.
The toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to fathead minnow (Pimephales promelas), channel catfish (Ictalurus punctatus), lake herring (Coregonus artedii), medaka (Oryzias latipes), white sucker (Catastomus commersoni), northern pike (Esox lucius), and zebrafish (Danio danio) were observed during early life-stage development after waterborne exposure of fertilized eggs. Species sensitivity based on TCDD-Cegg (TCDD concentration in eggs) was determined by effects observed over a 32-d period for all species except lake herring in which a 100-d period was used. Signs of TCDD toxicity, including edema, hemorrhaging, and craniofacial malformations were essentially identical to those observed in salmonids following TCDD egg exposure and preceded or accompanied mortality most often during the period from hatch through swim-up. The no-observed-effect concentrations and lowest-observed-effect concentrations, based on significant decreases in survival and growth as compared to the controls, ranged from 175 and 270 pg/g for lake herring to 424 and 2,000 pg/g for zebrafish, respectively. Shapes of concentration–response curves, expressed as TCDD-Cegg versus percent mortality, were similar for all species and were consistently steep suggesting that the mechanism of action of TCDD is the same among these species. The LCegg50s (concentrations in eggs causing 50% lethality to fish at test termination) ranged from 539 pg/g for the fathead minnow to 2,610 pg/g for zebrafish. Comparisons of LCegg50s indicate that the tested species were approximately 8 to 38 times less sensitive to TCDD than lake trout, the most sensitive species evaluated to date. When LCegg50s are normalized to the fraction lipid in eggs (LCegg,l50s), the risk to early life stage survival for the species tested ranges from 16- to 180-fold less than for lake trout.
Vertebrate embryos are particularly sensitive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Identification of tissues that are susceptible to the adverse effects of TCDD is requisite for understanding the embryo toxic effects of TCDD. The objective of the present study was to quantitate the temporal appearance of and dose dependence of apoptosis in TCDD-exposed medaka embryos (Oryzias latipes). A fluorescent-based DNA end-labeling assay provided a sensitive method for detection of TCDD-induced apoptosis in tissue sections of medaka embryos. Apoptotic cells were readily apparent in the medial yolk vein at all observed embryonic stages in TCDD-exposed embryos. Slope-comparison analysis indicated that TCDD-induced programmed cell death in the embryonic medial yolk vein was mechanistically linked to embryo mortality. These data are consistent with the hypothesis that vascular damage contributes to the acute embryo toxic effects of TCDD. However, as sublethal concentrations of dioxin-like compounds are more typical of environmental exposures, tissue damage was also assessed in medaka fry that were exposed to low doses of TCDD during embryonic development. Cell death was detected in gill and digestive tissues in visibly healthy medaka fry that had been exposed to low doses of TCDD during embryonic development. Increased expression of cytochrome P450 1A is a major biochemical consequence of TCDD exposure and is often used as a biomarker for exposure to dioxin-like compounds. Therefore, we compared the tissue distribution of TCDD-induced P450 1A expression and TCDD-induced programmed cell death. TCDD-induced programmed cell death co-localized with TCDD-induced P450 1A expression in both embryos and in visibly healthy post-hatch fry. Our results suggest that aberrant programmed cell death may be a suitable marker for exposure of feral organisms to dioxin-like compounds.
Hematopoiesis in the vertebrate is characterized by the induction of ventral mesoderm to form hematopoietic stem cells and the eventual differentiation of these progenitors to form the peripheral blood lineages. Several genes have been implicated in the differentiation and development of hematopoietic and vascular progenitor cells, yet our understanding of the discrete steps involved in the induction of these cells from the ventral mesoderm is still incomplete. One method of delineating these processes is based on the use of lower vertebrates. The zebrafish (Danio rerio) is an especially robust vertebrate system for both isolating and characterizing genes involved in these processes. Hematopoietic mutants have been generated with defects in many of the steps of both the primitive and definitive hematopoietic programs. Cloning of the genes that underlie these mutations should yield valuable details of hematopoiesis and may have therapeutic implications for bone marrow transplantation and stem cell gene therapy.
Female rhesus monkeys given a diet containing 500 ppt (t = 1012) 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 9 months became anaemic within 6 months and pancytopenic after 9 months of exposure. The marked thrombocytopenia was associated with widespread haemorrhage. Death occurred in five of the eight animals between months 7 and 12 of the experiment at total exposure levels of 2–3 μg TCDD/kg body weight. At autopsy, in addition to the extensive haemorrhage, there was a distinct hypocellularity of the bone marrow and lymph nodes. Hypertrophy, hyperplasia and metaplasia of the epithelium in the bronchial tree, bile ducts, pancreatic ducts, salivary-gland ducts and palpebral conjunctivae were observed. Squamous metaplasia and keratinization of the sebaceous glands and hair follicles were present in the skin. Death was attributed to complications from the severe pancytopenia.
Mice and guinea pigs were given a single oral dose of various chlorinated dibenzo-p-dioxins (CDD) to establish and compare the LD50 and clinical and pathologic manifestations of toxicity. It was apparent that the 2,3,7, and 8 positions must be chlorinated to achieve the greatest degree of toxicity. Additional chlorine atoms at an ortho position reduced toxicity but not nearly to the degree caused by deletion of a chlorine atom at one of the lateral positions. A decrease in body weight gain was the most sensitive clinical parameter and animals severely intoxicated showed a marked weight loss, especially guinea pigs. The median time to death at a LD50 was 17 to 20 days in guinea pigs and 22 to 25 days in mice. Doses 10 times greater did not markedly shorten this period. At the toxic dose the spectrum and severity of lesions and organ weight effects were similar for all homologs and isomers within the same animal species; however, there were interspecies differences. The thymus was greatly reduced in size in both animal species due to a reduction in the number of cortical lymphocytes. Significant macroscopic and histopathologic hepatic effects including porphyria were observed only in the mouse and were found at does levels well below the LD50. Hyperplasia of the transitional epithelium in the urinary tract was found in guinea pigs. There was a reduction of total serum protein in the mouse due to lower levels of α-globulin. Other lesions were generally interpreted to be a secondary response to debilitation.
Embryos mutant for the T gene, in mice, make insufficient mesoderm and fail to develop a notochord. We report the cloning and sequencing of the T gene in the zebrafish (Brachydanio rerio) and show the nuclear localization of the protein product. Both RNA and protein are found in cells of the germ ring, including enveloping layer cells, prior to and during gastrulation of zebrafish embryos. Nuclei of the yolk syncytial layer do not express Zf-T. High levels of expression are maintained throughout early development in the notochord, while in paraxial mesoderm cells the gene is turned off during gastrulation. Exposure of animal cap cells to activinA induces Zf-T expression, as does transplantation into the germ ring.
Previous studies have shown that high levels of cytochrome P450 can occur in cardiac microsomes of vertebrates [Mol. Pharmacol. 21:517-526, (1982)]. Here we identify the dominant cardiac P450 in the marine fish scup as P450E, a teleost representative of P450IA1, and we describe its restricted cellular localization in the heart. Treatment of scup with beta-naphthoflavone produced an unusually strong (10-fold) induction of spectrally measured P450 in cardiac microsomes, with specific content reaching levels (0.5 nmol/mg) similar to those induced in scup liver. Microsomal ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities, catalytic functions of scup P450E, were induced in parallel with P450 content. Similar induction was seen in both atrium and ventricle. Immunoblot analysis with monoclonal antibody 1-12-3, specific to scup P450E and other vertebrate P450IA1 proteins, showed that this hydrocarbon-inducible P450 is the dominant and possibly sole P450 form in heart microsomes of experimentally induced animals. Immunohistochemical analysis of scup heart sections (2-4-microns) with monoclonal antibody 1-12-3 revealed that P450E was detectable only in endothelial cells of the endocardium and of the coronary vasculature. A similar endothelial cell localization of the monoclonal antibody 1-12-3 epitope was observed in heart of rainbow trout, induced with beta-naphthoflavone, indicating a general nature for the endothelial localization of induced cardiac P450. Morphometric analysis showed that endothelium could constitute 8-9% of the volume of teleost heart, from which we calculate that P450IA1 could account for as much as 25% of the endothelial cell microsomal protein. Heart microsomes of untreated animals from contaminated environments also contained high levels of P450E, indicating that induction like that caused by beta-naphthoflavone could occur with chemicals in the environment. Strongly induced P450E (P450IA1) in endothelium could play a critical role in chemical-biological interactions involving xenobiotics affecting the vasculature of the heart or other organs.
Vertebrate hematopoietic stem cells are derived from vental mesoderm, which is postulated to migrate to both extra- and intraembryonic positions during gastrula and neurula stages. Extraembryonic migration has previously been documented, but the origin and migration of intraembryonic hematopoietic cells have not been visualized. The zebrafish and most other teleosts do not form yolk sac blood islands during early embryogenesis, but instead hematopoiesis occurs solely in a dorsal location known as the intermediate cell mass (IM) or Oellacher. In this report, we have isolated cDNAs encoding zebrafish homologs of the hematopoietic transcription factors GATA-1 and GATA-2 and have used these markers to determine that the IM is formed from mesodermal cells in a posterior-lateral position on the yolk syncytial layer of the gastrula yolk sac. Surprisingly, cells of the IM then migrate anteriorly through most of the body length prior to the onset of active circulation and exit onto the yolk sac. These findings support a hypothesis in which the hematopoietic program of vertebrates is established by variations in homologous migration pathways of extra- and intraembryonic progenitors.
This article reviews the present state of the art regarding the toxicokinetics and metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). The absorption, body distribution, and metabolism can vary greatly between species and also may depend on the congener and dose. In biota, the 2,3,7,8-substituted PCDDs and PCDFs are almost exclusively retained in all tissue types, preferably liver and fat. This selective tissue retention and bioaccumulation are caused by a reduced rate of biotransformation and subsequent elimination of congeners with chlorine substitution at the 2,3,7, and 8 positions. 2,3,7,8-Substituted PCDDs and PCDFs also have the greatest toxic and biological activity and affinity for the cytosolic arylhydrocarbon (Ah)-receptor protein. The parent compound is the causal agent for Ah-receptor-mediated toxic and biological effects, with metabolism and subsequent elimination of 2,3,7,8- substituted congeners representing a detoxification process. Congener-specific affinity of PCDDs and PCDFs for the Ah-receptor, the genetic events following receptor binding, and toxicokinetics are factors that contribute to the relative in vivo potency of an individual PCDD or PCDF in a given species. Limited human data indicate that marked species differences exist in the toxicokinetics of these compounds. Thus, human risk assessment for PCDDs and PCDFs needs to consider species-, congener-, and dose-specific toxicokinetic data. In addition, exposure to complex mixtures, including PCBs, has the potential to alter the toxicokinetics of individual compounds. These alterations in toxicokinetics may be involved in some of the nonadditive toxic or biological effects that are observed after exposure to mixtures of PCDDs or PCDFs with PCBs.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and structurally similar halogenated aromatic hydrocarbons cause a broad range of immunologic effects in experimental animals including decreased host resistance to infectious disease and suppressed humoral and cell-mediated immune responses. In the mouse, TCDD immunotoxicity has been shown to be an aryl hydrocarbon (Ah) receptor-dependent process. However, despite considerable research, the biochemical and molecular alterations that occur subsequent to Ah receptor activation that lead to altered immune reactivity remain to be elucidated. In addition to immune suppression, TCDD promotes inflammatory responses. This effect may result from an upregulation of the production of inflammatory cytokines such as interleukin-1 and tumor necrosis factor. Nonhuman primates exposed to TCDD show suppressed antibody responses and changes in lymphocyte subsets in the peripheral blood. The immunotoxic effects of TCDD in humans are poorly characterized, and few studies have examined the immune status of individuals with known, documented exposure to TCDD. It is important for laboratory research to focus on defining TCDD-sensitive immunologic biomarkers in animal models that can also be used in human subjects. Understanding the mechanisms that underlie species differences in TCDD immunotoxicity is also of critical importance for extrapolation of effects seen in laboratory animals to man.
I thank my many Eugene colleagues for delightful and insightful conversations about this work, for access to unpublished studies, and for critical comments on versions of the manuscript. Work in my lab is supported by grants from the National Institutes of Health (NS23915, HD22486).
Edema and cardiovascular dysfunction occur in vertebrates exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during early development. This study examined cytochrome P4501A (CYP1A) induction in endothelium and its possible association with mortality due to the edema and vascular effects of TCDD in lake trout early life stages. Lake trout (Salvelinus namaycush) eggs were injected at 24-50 hr postfertilization with 0.2 microl of 50 mM phosphatidylcholine liposomes or liposomes containing TCDD to give seven doses ranging from 11 to 176 pg TCDD/g egg. Doses of TCDD greater than 44 pg/g egg elicited hemorrhages; yolk sac, pericardial, and meningial edema; craniofacial malformations; regional ischemia; growth retardation; and mortality at the sac fry stage of development. Expression of CYP1A was assessed at four developmental stages, by immunohistochemical analysis of serial sections of individual fish with monoclonal antibody 1-12-3 to teleost CYP1A. CYP1A staining occurred in endothelial cells of many organs of TCDD-exposed but not vehicle-exposed embryos at 1 week prehatch and sac fry at 2 weeks posthatch. Earlier developmental stages examined were negative for CYP1A expression at any dose of TCDD. The strongest response occurred in sac fry at TCDD doses greater than 88 pg TCDD/g egg but was detected at doses as low as 22 pg TCDD/g egg. CYP1A staining in endothelium appeared at lower doses and was stronger than that in other cell types, in both prehatch embryos and posthatch sac fry. Thus, the vascular system is a major initial site affected by TCDD in lake trout early life stages, and the vascular endothelium is a cell type uniquely sensitive to induction of CYP1A in these developing animals. Based on an index of immunohistochemical staining of CYP1A, endothelial CYP1A induction in sac fry by TCDD occurred with an ED50 of 64-69 pg TCDD/g egg, similar to the dose-response for mortality occurring during the sac fry stage of development (LD50 = 47 pg TCDD/g egg). The correlations seen here suggest that CYP1A or aryl hydrocarbon receptor (AhR) in the endothelium may be linked to early lesions that result in TCDD-induced vascular derangements leading to yolk sac, pericardial, and meningial edema that is associated with lake trout sac fry mortality, but the precise mechanism remains to be determined.
We have analyzed the possible role of the aryl-hydrocarbon receptor (AHR) in the aging process of mice using a homozygous null mouse (Ahr-/-) line as a model. We studied 52 male and female Ahr-/- mice aged from 6-13 months. Forty-six percent died or were ill by 13 months of age. Ahr-/- mice developed age-related lesions in several organs, some of which were apparent after only 9 months of age. Cardiovascular alterations included cardiomyopathy (100%) with hypertrophy and focal fibrosis. Vascular hypertrophy and mild fibrosis were found in the portal areas of the liver (81%), and vascular hypertrophy and mineralization were common in the uterus (70%). Gastric hyperplasia that progressed with age into polyps was evident in the pylorus of 71% of the mice over 9 months of age. Ahr-/- mice had T-cell deficiency in their spleens but not in other lymphoid organs. The immune system deficiency described previously could be the origin for the rectal prolapse found in 48% of the null mice, associated with Helicobacter hepaticus infection. In the dorsal skin (53% incidence), severe, localized, interfollicular and follicular epidermal hyperplasia, with hyperkeratosis and acanthosis, and marked dermal fibrosis, associated with the presence of anagenic hair follicles, were also evident. None of these lesions were found in 42 control (Ahr +/+ or +/-) mice of similar ages. These observations suggest that the AHR protein, in the absence of an apparent exogenous (xenobiotic) ligand, plays an important role in physiology and homeostasis in major organs in mice, and further supports an evolutionary conserved role for this transcription factor.
Recently, molecular markers such as recombination activating genes (RAG), terminal deoxynucleotidyl transferase (TdT), stem cell leukemia hematopoietic transcription factor (SCL), Ikaros and gata-binding protein (Gata)-family members have been isolated and characterized from key lower vertebrates, adding to our growing knowledge of lymphopoiesis in ectotherms. In all gnathostomes there appear to be two main embryonic locations derived from the early mesoderm, both intra- and extraembryonic, which contribute to primitive and definitive hematopoiesis based upon their differential expression of SCL, Gata-1, Gata-2 and myeloblastosis oncogene (c-myb). In teleosts, a unique intraembryonic location for hematopoietic stem cells termed the intermediate cell mass (ICM) of Oellacher appears to be responsible for primitive or definitive hematopoiesis depending upon the species being investigated. In Xenopus, elegant grafting studies in combination with specific molecular markers has led to a better definition of the roles that ventral blood islands and dorsal lateral plate play in amphibian hematopoiesis, that of primitive and definitive lymphopoiesis. After the early embryonic contribution to hematopoiesis, specialized tissues must assume the role of providing the proper microenvironment for T and B-lymphocyte development from progenitor stem cells. In all gnathostomes, the thymus is the major site for T-cell maturation as evidenced by strong expression of developmental markers such as Ikaros, Rag and TdT plus expression of T-cell specific markers such as T-cell receptor beta and lck. In this respect, several zebrafish mutants have provided new insights on the development of the thymopoietic environment. On the other hand, the sites for B-cell lymphopoiesis are less clear among the lower vertebrates. In elasmobranchs, the spleen, Leydig's organ and the spiral valve may all contribute to B-cell development, although pre-B cells have yet to be fully addressed in fish. In teleosts, the kidney is undeniably the major source of B-cell development based upon functional, cellular and molecular indices. Amphibians appear to use several different sites (spleen, bone marrow and/or kidney) depending upon the species in question.
The aryl hydrocarbon receptor (AhR) mediates the toxicity of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds in vertebrates. To further establish zebrafish as a vertebrate model to study the molecular mechanism of TCDD toxicity, we have isolated and characterized the cDNA encoding the zebrafish aryl hydrocarbon receptor (zfAhR2). Analysis of the deduced protein sequence revealed the 1027 amino acid protein is approximately 200 amino acids longer than previously isolated receptors. zfAhR2 is homologous to previously cloned PAS proteins within the basic helix-loop-helix and PAS domains. The C-terminal domain of zfAhR2 diverges from the mammalian AhR at position 420, and does not contain a Q-rich domain. zfAhR2 mRNA is first detected by Northern blot analysis at 24 h post fertilization, and expression increases throughout early development. Treatment of zebrafish embryos and zebrafish liver cells with graded doses of TCDD results in a dose-dependent increase in zfAhR2 mRNA. The time course for zfAhR2 and cytochrome P4501A mRNA induction by TCDD are similar. In vitro produced zfAhR2 protein dimerizes with the rainbow trout aryl hydrocarbon receptor nuclear translocator (rtARNTb) and binds dioxin response elements derived from the rainbow trout CYP1A gene. Finally, transient coexpression of zfAhR2 and rtARNTb in COS-7 cells results in a TCDD dose-related increase in transcription driven by the rainbow trout CYP1A promoter and enhancer.
The study of blood has often defined paradigms that are relevant to the biology of other vertebrate organ systems. As examples, stem cell physiology and the structure of the membrane cytoskeleton were first described in hematopoietic cells. Much of the reason for these successes resides in the ease with which blood cells can be isolated and manipulated in vitro. The cell biology of hematopoiesis can also be illuminated by the study of human disease states such as anemia, immunodeficiency, and leukemia. The sequential development of the blood system in vertebrates is characterized by ventral mesoderm induction, hematopoietic stem cell specification, and subsequent cell lineage differentiation. Some of the key regulatory steps in this process have been uncovered by studies in mouse, chicken, and Xenopus. More recently, the genetics of the zebrafish (Danio rerio) have been employed to define novel points of regulation of the hematopoietic program. In this review, we describe the advantages of the zebrafish system for the study of blood cell development and the initial success of the system in this pursuit. The striking similarity of zebrafish mutant phenotypes and human diseases emphasizes the utility of this model system for elucidating pathophysiologic mechanisms. New screens for lineage-specific mutations are beginning, and the availability of transgenics promises a better understanding of lineage-specific gene expression. The infrastructure of the zebrafish system is growing with an NIH-directed genome initiative, providing a detailed map of the zebrafish genome and an increasing number of candidate genes for the mutations. The zebrafish is poised to contribute greatly to our understanding of normal and disease-related hematopoiesis.
Treatment of adult C57BL6J mice with tetrachlorodibenzo-p-dioxin (TCDD) elicits altered bone marrow hemopoietic cellular potentials and markedly reduced T-lymphoid-reconstituting activity. The latter has been hypothesized to play a role in TCDD-induced thymic atrophy. To investigate cellular targets responsible for reduced prothymocyte capacity, bone marrow cells from TCDD-treated C57BL/6J mice were assessed for hemopoietic alterations within the lineage-negative (lin-) compartment by the examination of Sca-1 and c-Kit levels. Lin- hemopoietic cells from C57BL/6J mice, treated with 30 microg/kg of TCDD, were assessed for phenotypic alterations following 24 h through 31 days. The responses of lin- cells to TCDD doses ranging from 0.3 to 30 microg/kg were also assessed at 2 days following TCDD treatment. The data reveal increases in the number of bone marrow lin- Sca-1+ c-Kit+ cells, relative to control, over 24 h through 31 days following treatment, as well as dose-dependent increases in this population when examined at 2 days. Increases in lin- Sca-1+ c-Kit- cells occurred on a more transient basis and were also dependent upon TCDD dose. These data suggest that proliferation and/or differentiation processes of hemopoietic stem cells are affected by TCDD and that these effects contribute to a reduced capacity of bone marrow to generate pro-T lymphocytes.
Lake trout embryos exposed to [(3)H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) manifest toxicity after hatching by subcutaneous edema of the yolk sac, pericardial edema, meningeal edema, subcutaneous hemorrhages, and a marked congestion of blood flow in various vascular beds culminating in death. Our objective was to determine if this syndrome was associated temporally with morphologic lesions in the vascular endothelium, increased vascular permeability, and cytochrome P4501A (CYP1A) mRNA induction. Lake trout embryos exposed as fertilized eggs to TCDD were found to exhibit marked reductions in perfusion of the peripheral vasculature during the early sac fry stage of development (stage F(1)9), which consistently preceded other gross lesions and mortality observed later in sac fry development (stage F(2)10). This reduction in blood flow was manifested as severe capillary congestion and hemoconcentration in certain vascular beds. Transmission electron microscopic (TEM) examination of endothelial cells in these vascular beds failed to reveal cellular necrosis at hatching (stage E(5)8) and throughout sac fry development (stages F(1)9-F(2)10). Rather, only subtle ultrastructural changes in endothelial cells were found consisting of increased vacuolation, separation of intercellular junctions, and cytoplasmic blebbing, consistent with the TCDD dose and time course for developmental cardiovascular toxicity, which began to manifest itself in some embryos approximately 1 week prior to hatching (E(5)8). To assess permeability of yolk sac vasculature to certain constituents in blood, sac fry (stage F(2)10) were analyzed for the presence of plasma proteins, granulocytes, and serum creatine kinase activity in yolk sac subcutaneous edema fluid from control and TCDD-exposed treatment groups. TCDD dose- and time-related increases in yolk sac edema volume, plasma protein content of edema fluid, granulocyte concentration, and creatine kinase activity in the fluid were observed in midstage and late stage of sac fry development (stage F(2)10). Thus, yolk sac subcutaneous edema fluid is an ultrafiltrate of blood and results from increased vascular permeability. In contrast to the changes in vascular blood flow and permeability induced by TCDD during stages F(1)9 and F(2)10 of sac fry development, respectively, CYP1A mRNA levels were induced by TCDD as early as the 10-somite embryo (stage E(2)5). TCDD also caused a dose-related increase in CYP1A mRNA levels in sac fry at hatching (stage E(5)8) and throughout sac fry development (stages F(1)9-F(2)10). We conclude that subtle, ultrastructural changes in vascular endothelial cells consistently precede increases in vascular permeability and sac fry mortality; however, induction of CYP1A mRNA occurs prior to any observable morphological lesions, changes in vascular permeability, or sac fry mortality.
We report the generation and characterization of transgenic mouse and zebrafish expressing green fluorescent protein (GFP) specifically in vascular endothelial cells in a relatively uniform fashion. These reporter lines exhibit fluorescent vessels in developing embryos and throughout adulthood, allowing visualization of the general vascular patterns with single cell resolution. Furthermore, we show the ability to purify endothelial cells from whole embryos and adult organs by a single step fluorescence activated cell sorting. We expect that these transgenic reporters will be useful tools for imaging vascular morphogenesis, global gene expression profile analysis of endothelial cells, and high throughput screening for vascular mutations.
Compar-ative toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to seven fresh-water species during early life-stage development
- Elonen Ge
- Rl Sphear
- Holcombe
- Gw
- Johnson
Elonen GE, Sphear RL, Holcombe GW, Johnson RD. 1998. Compar-ative toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to seven fresh-water species during early life-stage development. Environ Toxicol Chem 17:472– 483.
SCL/Tal-1 transcription factor acts downstream of cloche to specify hematopoietic and vascular progenitors in zebrafish
- Liao Ec
- Oates Bh Ac Paw
- Pratt
- Sj
- Postlethwait Jh
- Zon
- Li
Liao EC, Paw BH, Oates AC, Pratt SJ, Postlethwait JH, Zon LI. 1998. SCL/Tal-1 transcription factor acts downstream of cloche to specify hematopoietic and vascular progenitors in zebrafish. Genes Dev 12:621– 626.
Use of the zebrafish (Danio rerio) to define hematopoiesis
- Bahary N
- Zon LI
Universal GFP reporter for the study of vascular development
- Motoike T
- Loughna S
- Perens E
- Roman BL
- Liao W
- Chau TC
- Richardson CD
- Kawate T
- Kuno J
- Weinstein BM
- Stainier DY
- Sato TN
Comparative toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to seven freshwater species during early life-stage development
- G E Elonen
- R L Sphear
- G W Holcombe
- R D Johnson
Elonen GE, Sphear RL, Holcombe GW, Johnson RD. 1998. Comparative toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to seven freshwater species during early life-stage development. Environ Toxicol
Chem 17:472-483.
- Kerkvliet
- Motoike