* Department of Toxicology, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu 069-8501, Japan;
Veterinary Internal Medicine II, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu 069-8501, Japan;
Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan; and
School of Pharmacy, University of Wisconsin, Madison, Wisconsin 537052222
Received May 4, 2001; accepted October 17, 2001
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ABSTRACT |
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Key Words: Ah receptor (AhR); 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); zebrafish; embryo; developmental toxicity; craniofacial; jaw; cardiovascular; blood flow; cartilage.
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INTRODUCTION |
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Zebrafish (Danio rerio) has been extensively used in developmental biology for 10 years. They have several advantages as a vertebrate toxicological model. They produce many embryos of high quality. They develop rapidly and hatch as early as 2 days after fertilization. They have a transparent body adequate for the observation of internal organs by conventional microscopy in early stages of development. They are applicable for both forward and reverse genetics. Several thousand mutant lines are established and available (Haffter et al., 1996). Zebrafish embryos have further advantages as a toxicological model because there is a wealth of information on their genetics and developmental biology. Also zebrafish embryos are responsive to TCDD developmental toxicity (Henry et al., 1997
) and the aryl hydrocarbon receptor (AhR) and its dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT) have been cloned and functionally characterized in this species (Tanguay et al., 1999
, 2000
, 2001
). In the present study, we examined, by quantitative measurement, the lower jaw growth and RBC perfusion rate as an index of local blood flow in zebrafish embryos exposed to TCDD.
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MATERIALS AND METHODS |
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Zebrafish embryos and waterborne TCDD exposure.
According to Westerfield (1993), fertilized embryos were obtained from natural mating of adult zebrafish (AB line) originally derived from the University of Oregon, Eugene, OR. Adult and juvenile fish were maintained at 28.5°C in a 14-h light and 10-h dark cycle. Newly fertilized embryos were exposed to TCDD dissolved in 1/3 Ringer solution (Zebrafish Ringer solution, ZR solution; Dong et al., 2001) in petri dishes at 28.5°C. Usually 10 embryos were kept in 3 ml of ZR solution in a 3-cm plastic petri dish (Falcon). However, for assessment of mortality, embryos were kept individually in 1 ml of ZR solution in an individual well of a 24-multiwell dish (Falcon). This was done to exclude potential harmful effects of decaying dead embryos on those that were still alive. Unless otherwise noted, embryos were continuously exposed to the waterborne vehicle (0.1% DMSO) or to graded concentrations of waterborne TCDD (0.11.0 ppb). The exposures started at 24-h postfertilization (hpf) and continued until the time of observation. The composition of ZR solution was as follows: 38.7 mM NaCl, 1.0 mM KCl, 1.7 mM HEPES (pH 7.2), and 2.4 mM CaCl2. Three µl of TCDD stock solution in dimethylsulfoxide (DMSO) was directly added to a 3-cm petri dish containing 3 ml of ZR solution. Thus, the final concentration of the DMSO vehicle was usually 0.1% regardless of TCDD concentration.
Measurement of lower jaw growth.
Live embryos from 48 to 120 hpf were observed microscopically in 3% carboxymethyl cellulose sodium salt/ZR solution after being anesthetized with FA100 (Tanabe, Japan) or MS 222 (Sigma). The length of the lower jaw was determined with an oculometer. We used the anterior edge of the eye as a reference point.
Alcian-blue staining.
Zebrafish embryos were stained with alcian blue according to the method of Kelly and Bryden (1983). 10% Neutral formalin-fixed embryos were stained with 0.1% alcian blue 8GX/80% ethanol/20% acetic acid for 6 h. After a series of washes with 75% and 50% ethanol/PBS each for 1 h, embryos were incubated with PBS overnight. For clarification, embryos were treated with 1% KOH/3% H2O2 for 20 h, followed by digestion with 0.05% trypsin/saturated tetraborate for 1 h. Stained embryos were preserved and observed in 80% glycerol solution.
In situ hybridization.
Whole mount in situ hybridization was carried out according to Barth and Wilson (1995). Four percent paraformaldehyde (PFA)-fixed embryos were treated with proteinase K (Sigma) in PBS with 0.1% Tween (PTw). After incubation with hybridization buffer (Hyb [-]) containing 50% formamide, 5x SSC, 2 mg/ml Torula RNA (Sigma), and 200 µg/ml heparin (prehybridization) (Hyb [+]), embryos were hybridized with antisense probes of zebrafish goosecoid (gsc) or zfAhR2 at 65°C overnight. To obtain the zfAhR2 probe, cDNA was cloned by PCR according to Tanguay et al. (1999). cDNA for gsc was a gift from S.W. Wilson. Following hybridization, embryos were washed with 2x SSC and 0.2x SSC for 30 min, twice, at 65°C, respectively. After blocking with a blocking buffer containing 2% blocking reagent (Roche, Germany) in 100 mM maleic acid (pH 7.5) and 150 mM NaCl, embryos were incubated with 1:4000 diluted anti-DIG antibody conjugated with alkaline phosphatase (Roche) at 4°C overnight. The color reaction was carried out by incubation in BM-purple substrate or Fast Red (Roche) after equilibration with NTMT buffer (100 mM NaCl, 100 mM TrisHCl, pH 9.5, and 50 mM MgCl2) at room temperature for 15 min. In the case of zfAhR2, 0.003% phenylthiourea (Sigma) was included in ZR solution from 24 hpf to prevent pigmentation of the embryos. Pigments of embryos with gsc signals were bleached by 6% H2O2 for 2 h. After PFA fixation, the stained embryos were cleared in 70% glycerol and used for observation. In some experiments, stained embryos were sectioned after embedding in paraffin.
Measurement of RBC perfusion rate.
As an index of local circulation, RBC perfusion rate was evaluated by time-lapse recording using a digital video camera (DVL-700, Victor, Japan) connected to a CCD camera (MKC-385, Ikegami, Japan). Embryos were observed in 200 µl of 3% carboxymethyl cellulose/ZR solution in a bath on the stage of an inverted microscope (Olympus, Japan). The temperature was maintained at 28.5°C with a PDMI-2 Micro-incubator (Medical Systems). Nomenclature for the vessels of zebrafish embryos was that of Weinstein et al. (http://www.dir.nichd.nih.gov/lmg/uvo/WEINSLAB.html).
Statistics.
Results are presented as mean ± SEM. Significance was determined by one-way ANOVA followed by the Bonferroni/Dunn test (p < 0.05).
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RESULTS |
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Figure 1 shows representative photographs of live embryos treated with vehicle (control) or TCDD. Effects of TCDD are shown on total length of the body in Figure 2A
and on lower jaw length in Figure 2B
, respectively. Total body length was not markedly affected by TCDD. TCDD at an exposure level of >0.3 ppb caused a slight but significant reduction in total body length at 7284 hpf, whereas TCDD at 0.1 ppb did not have any effect (Fig. 2A
). The jaw of zebrafish embryos appeared from 48 hpf and grew gradually to reach the anterior edge of the eye after around 72 hpf (protruding mouth; Figs. 1 and 2
). Positive value means that the mouth is placed in front of the anterior edge of the eye and negative value means that the mouth is placed behind the anterior edge of the eye (Fig. 2
, inset photos). Since TCDD exposure caused a slight reduction in total body length, each value for lower jaw length (Fig. 2B
) is indicated as a percentage of total body length. In examining Fig. 2B
, it can be seen that TCDD reduced lower jaw growth as early as 60 hpf. This inhibitory effect was dependent on the waterborne concentration of TCDD. TCDD concentrations of 0.3, 0.5, and 1.0 ppb exerted significant effects that were first detected at 84, 72, and 60 hpf, respectively. On the other hand, 0.1 ppb TCDD did not have any effect until 120 hpf (Fig. 2B
). These results suggest that the effects of TCDD were lower jaw-specific and not merely secondary to a reduction in embryo growth.
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Local Circulation Failure Induced by TCDD
The reduction of RBC perfusion rate in various vascular beds was consistently observed in TCDD-exposed embryos of many fish species including zebrafish (Henry et al., 1997; Hornung et al., 1999
; Walker and Peterson, 1994
). However, in all cases these have been subjective measurements of the RBC perfusion rate that was scored by the observer. In the present study, local circulation was quantitatively evaluated by counting the number of RBCs passing through certain vessels with time-lapse recording. Before RBC perfusion rate was measured, we assessed effects of TCDD exposure on heart rate and pericardial edema. Regardless of the concentration of TCDD used (0.31 ppb), heart rate did not change until 96 hpf when most of the embryos showed severe pericardial edema (Fig. 1E
). In control embryos, pericardial edema was not observed. After treatment with 1 ppb TCDD, pericardial edema was first observed in 35% of the embryos at 60 hpf and increased gradually to reach almost 100% of the embryos at 96 hpf (Fig. 6
). A significant finding was that the heart was flattened under pressure of the extensive pericardial edema by 96 hpf.
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DISCUSSION |
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Expression of AhR and CYP1A
AhR mRNAs were strongly expressed in the lower jaw primordia during its extensive growth. The AhR transcript expression area covers Meckel's cartilage and can be detected as early as 30 hpf, which is before the general circulation failure that occurs in TCDD-exposed embryos. Therefore, TCDD may act directly to impair chondrogenesis in lower jaw primordia via an AhR-dependent mechanism that is independent of circulation failure. AhR expression in the lower jaw primordia has also been reported in mammals including human (Abbott et al., 1994, 1998
). Also CYP1A mRNA and protein were expressed constitutively in the lower jaw primordia of control fathead minnow and other fish species including zebrafish, and was markedly induced by TCDD in all of these species (Iwata and Stegeman, 2000
; Teraoka et al., unpublished observations).
Edema and Local Circulatory Failure
The mechanisms involved in TCDD cardiovascular toxicity in the zebrafish embryo remain to be determined. Pericardial edema occurred in 35% of TCDD-exposed embryos at 60 hpf, but at this time RBC perfusion rate in the vessels examined in the present study did not change, and this also appears to include the cardinal vessels. A marked reduction of RBC perfusion rate was first detected at 72 hpf in vessels of the trunk. However, whether the early edema is a primary event or is secondary to reduced trunk blood flow remains to be determined. The magnitude of RBC perfusion rate reduction was different for different vessels, but the reason for this differential effect of TCDD on blood flow at 72 hpf is unknown. It may be that TCDD exposure produces differential effects on endothelial cells in different blood vessels of the zebrafish embryo. Cantrell et al. (1996, 1998) reported a possible relationship between CYP1A expression and TUNEL signals in endothelial cells in the yolk sac vein of medaka embryos exposed to TCDD. However, Guiney et al. (2000) failed to find evidence of enhanced apoptosis of vascular endothelial cells in lake trout embryos but found instead a slight gap between endothelial cells, suggesting an increase in vascular permeability. In cultured porcine endothelial cells, exposure to the AhR agonist PCB 77 augmented albumin permeability, but only produced apoptosis when endothelial cell cultures were treated with PCB 77 and a glutathione synthesis inhibitor, buthionin-sulphoxamine (Slim et al., 2000; Toborek et al., 1995
). In TCDD-exposed zebrafish embryos, the cause of the severely reduced RBC perfusion rate that occurs in all vessels later in development at 96 hpf, is probably related to decreased cardiac output caused by the progressively extensive pericardial edema that severely flattens the heart. A reduction of circulating RBCs in TCDD-exposed zebrafish and Xenopus embryos has also been reported to occur and would further enhance the hypoxic insult caused by the circulatory failure at 96 hpf (Belair et al., 2001
; Sakamoto et al., 1997
). The progressive circulatory failure in zebrafish embryos exposed to TCDD could also be due to an effect on the heart. In the chick embryo heart, AhR and ARNT are expressed (Walker et al., 1997
) and CYP1A is induced by AhR agonists (Gannon et al., 2000
). TCDD causes cardiac malformations in the chick embryo (Cheung et al., 1981
; Walker et al., 1997
; Walker and Catron, 2000
) as well as functional changes in cardiac myocytes of TCDD-treated chick embryos (Canga et al., 1988
). Therefore, extensive studies of TCDD effects on the embryo heart are also needed to understand the mechanism of the TCDD-induced circulatory failure in zebrafish, especially for the later stage.
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ACKNOWLEDGMENTS |
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NOTES |
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