2,3,7,8-Tetrachlorodibenzo-p-dioxin Toxicity in the Zebrafish Embryo: Local Circulation Failure in the Dorsal Midbrain Is Associated with Increased Apoptosis

Wu Dong*, Hiroki Teraoka*,1, Koji Yamazaki*, Shusaku Tsukiyama*, Sumiko Imani*, Tomohiro Imagawa{dagger}, John J. Stegeman{ddagger}, Richard E. Peterson§ and T. Hiraga*

* Department of Toxicology, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu 069-8501, Japan; {dagger} Department of Veterinary Anatomy, University of Tottori, Japan; {ddagger} Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts; and § School of Pharmacy and Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin

Received March 7, 2002; accepted May 15, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on local circulation and apoptosis in the midbrain were investigated in zebrafish (Danio rerio) embryos during early development. Embryos were exposed to TCDD from 24 h post fertilization (hpf) until observation, in water maintained at 28.5°C. TCDD decreased blood flow in the mesencephalic vein, the only vessel perfusing the dorsal midbrain of the embryo. At 50 hpf, blood flow was maximally reduced in this vessel and gradually returned to the control level at 60 hpf. In contrast, blood flows in the trunk and in other vessels of the head of the embryo did not significantly change until 72 hpf. Furthermore, TCDD exposure caused apoptosis in the midbrain at 60 hpf, and the TCDD dose response relationship for this effect was similar to that for reduced blood flow in the mesencephalic vein at 50 hpf. The effects of TCDD on apoptosis in the midbrain, but not on blood flow, were abolished by Z-VAD-FMK, a general caspase inhibitor. TCDD effects on both endpoints were mimicked by ß-naphthoflavone (BNF), an aryl hydrocarbon receptor (AHR) agonist, and almost abolished by concomitant exposure to TCDD and {alpha}-naphthoflavone (ANF), an AHR antagonist. Concomitant exposure to TCDD and either an inhibitor of cytochrome P450 (CYP) (SKF525A or miconazole) or an antioxidant (N-acetylcysteine or ascorbic acid) inhibited these effects of TCDD. The incidence of apoptosis in the midbrain was inversely related to blood flow in this brain region following these various treatments and graded TCDD exposure concentrations (r = –0.91). The same range of TCDD exposure concentrations that reduced blood flow and increased apoptosis in the midbrain greatly enhanced CYP1A mRNA expression and immunoreactivity at 50 hpf in endothelial cells of blood vessels including the mesencephalic vein and the heart, but not the brain parenchyma. Taken together, these results suggest that TCDD induces apoptosis in the midbrain of the zebrafish embryo secondary to local circulation failure, which could be related to AHR activation, induction of CYP1A, and oxidative stress.

Key Words: apoptosis; CYP1A; circulation failure; cytochrome P4501A; developmental toxicity; TCDD; dioxin; endothelial cell; midbrain; zebrafish embryo; aryl hydrocarbon receptor; oxidative stress; mesencephalic vein.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure of fish larvae to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a reduction in peripheral circulation, edema, craniofacial malformation, and growth retardation, and culminating in mortality (Elonen et al., 1998Go; Henry et al., 1997Go; Walker and Peterson, 1994Go). These signs of toxicity are essentially identical to blue sac disease in rainbow trout larvae (Spitsbergen et al., 1990Go) and are of ecological concern. Lake trout larvae, for example, are very sensitive to TCDD-induced mortality (Spitsbergen et al., 1990Go; Walker et al., 1991Go), and it has been estimated that concentrations of TCDD equivalents in wild lake trout eggs, sufficient to cause 100% mortality, have occurred in the past in Lake Ontario (Cook et al., 1997Go).

An important outcome of the early research on TCDD developmental toxicity in fish larvae was identification of the cardiovascular system as a site of action, a site of strong induction of CYP1A (Stegeman et al., 1989Go). It was found that TCDD-induced mortality was preceded by circulatory failure in larvae of various fish species (Elonen et al., 1998Go; Henry et al., 1997Go; Spitsbergen et al., 1990Go; Walker and Peterson, 1994Go). In lake trout larvae, the TCDD dose-response curve for induction of cytochrome P4501A (CYP1A) in the vascular endothelium was essentially identical to that for larval mortality (Guiney et al., 1997Go). In rainbow trout larvae, the signs of TCDD-induced blue sac syndrome were associated with a decrease in heart size as well as a decrease in peripheral blood flow (Hornung et al., 1999Go). In the medaka embryo, TCDD caused both CYP1A induction and apoptosis in the vascular endothelium of the yolk sac, and the two effects were blocked by an antioxidant (Cantrell et al., 1996Go, 1998Go). Finally, results in TCDD-exposed zebrafish and lake trout larvae also suggest that vascular endothelial function is disrupted (Guiney et al., 2000Go; Henry et al., 1997Go).

It is notable that these findings are consistent with the association between TCDD exposure and cardiovascular disease in humans (Calvert et al., 1998Go; Flesch-Janys et al., 1995Go), a role for the aryl hydrocarbon receptor (AHR) in vascular remodeling during fetal development in mice (Lahvis et al., 2000Go), and TCDD-like AHR agonists acting directly on endothelial cells in primary culture. With respect to the latter, PCB 77 increases CYP1A, oxidative stress, and permeability of primary porcine aorta endothelial cells (Stegeman et al., 1995Go; Toborek et al., 1995Go), and this TCDD-like AHR agonist also activates caspase-3 and causes apoptosis under oxidative stress caused by pretreatment with a glutathione synthesis inhibitor (Slim et al., 2000Go).

Adverse effects of TCDD on the central nervous system (CNS) of fish larvae have only begun to be investigated. Also the brain and spinal cord are critically dependent on the cardiovascular system for adequate perfusion of blood. If cardiovascular function is disrupted, CNS function will be impaired secondarily. In TCDD-exposed lake trout larvae, cellular necrosis of the brain and spinal cord was observed immediately prior to death. Histopathologic evaluation revealed that the necrosis was secondary to a reduction in blood flow, but the magnitude of cerebral ischemia was not quantitated (Spitsbergen et al., 1990Go). TCDD also causes injury to rat hippocampal neurons in culture, which is associated with an increase in intracellular calcium (Hanneman et al., 1996Go). We discovered that TCDD induces apoptotic cell death in the dorsal midbrain of early zebrafish embryos (Dong et al., 2001Go) and a similar observation has been reported in Fundulus heteroclitus embryos following TCDD exposure (Toomey et al., 2001Go). In zebrafish embryos, the effect of TCDD was mimicked by the AHR agonist, ß-naphthoflavone (BNF), and was inhibited by the AHR antagonist, {alpha}-naphthoflavone (ANF), suggesting that TCDD-induced apoptosis in the dorsal midbrain of zebrafish larvae is an AHR-dependent response (Dong et al., 2001Go).

The objective of the present study was to determine if TCDD exposure causes circulation failure in the dorsal midbrain of the zebrafish embryo and, if so, if it plays a role in the increase in apoptosis caused by TCDD. The central nervous system of zebrafish larvae is expected to respond to TCDD, because AHR2 and ARNT2 messages are detected in this tissue (Andreasen et al., 2002bGo). Herein, we show that TCDD induces local circulation failure followed by apoptosis in the zebrafish embryo dorsal midbrain. The two responses appear associated. Both are caused by exposure to AHR agonists and are prevented by pretreatment of TCDD-exposed zebrafish embryos with either an AHR antagonist or inhibitors of CYP1A enzyme activity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was obtained from Cambridge Isotope Laboratories (98% purity, Andover, MA). SKF-525A (alpha-phenyl-alpha-propylbenzeneacetic acid 2-[diethylamino]ethylester) and Z-VAD-FMK (Z-Val-Ala-Asp (OMe)-CH2F) were purchased from Sigma (St. Louis, MO) and the Peptide Institute (Japan), respectively. Other chemicals were obtained from Wako Pure Chemical (Japan).

Zebrafish eggs and TCDD treatment.
Fertilized eggs were obtained from YJR natural mating of adult zebrafish (AB line) in our laboratory, carried out according to the Zebrafish Book (Westerfield, 1995Go). Adult fish and embryos were maintained at 28.5°C with a lighting schedule of 14 h light and 10 h dark. Eggs were collected within 1 h of spawning, rinsed, and placed into a clean petri dish. Within 24 h of spawning, newly fertilized eggs were exposed to either the TCDD vehicle, dimethyl sulfoxide (DMSO, usually 0.1%), or an apparent concentration of waterborne TCDD of 0.1, 0.3, 0.5, or 1.0 parts per billion (ppb) dissolved in 0.1% DMSO in 3 ml of Zebrafish Ringer‘s solution (38.7 mM NaCl, 1.0 mM KCl, 1.7 mM HEPES-NaOH pH 7.2, 2.4 mM CaCl2) in 3-cm petri dishes for the duration of the experiment (n = 10 embryos/dish).

TUNEL staining.
At 60 h post fertilization (hpf), embryos were fixed in 10% formalin for 24 h, followed by the conventional procedure for TUNEL staining as described previously (Dong et al., 2001Go; Gavrieli et al., 1992Go). Positive TUNEL signals were detected with ABC KIT (Elite, VECTOR) and counter-stained with methyl green. TUNEL-positive signals in both forebrain and midbrain were counted in all serial sections of each vehicle and TCDD-exposed embryo examined, because there was not a clear border between them (Dong et al., 2001Go).

Blood flow.
Blood flow in the mesencephalic vein, which perfuses the dorsal midbrain of the zebrafish embryo, was evaluated by time-lapse recording using a digital-video camera, as described previously (Teraoka et al., 2002Go). Embryos were suspended in 200 µl of 3% carboxymethyl cellulose/Zebrafish Ringer solution in a hand-made plastic bath mounted on the stage of an inverted microscope (Olympus). Temperature of the suspension solution was maintained at 28.5 °C with a PDMI-2 Micro-Incubator (Medical Systems). The nomenclature used to name vessels of the zebrafish embryo was that of Isogai et al. (2001).

Antibody staining.
Immunohistochemistry and immune electron microscopy were performed using a monoclonal antibody specific for fish CYP1A (Mab 1–12–3, Park et al., 1986Go). For immunohistochemistry, paraffin sections of vehicle and TCDD-exposed zebrafish embryos were incubated with phosphate-buffered saline with 0.5% triton X-100 and 1% horse serum (PBTR) for 30 min after proteinase-K treatment. The embryos were incubated with anti-CYP1A mouse monoclonal antibody (Mab-1–12–3) (x24,000) overnight at 4°C, followed by horseradish peroxidase-conjugated anti-mouse IgG antibody after washing with PBTR. Signals were detected with ABC KIT. For immune electron microscopy, embryos were fixed in a solution of 2% paraformaldehyde and 1% glutaraldehyde for 2 h or overnight at 4°C (Imagawa et al., 1991Go). After dehydration with an alcohol series, embryos were embedded in Lowicryl K4M (Chemische Werke Lowi, Germany) at –20°C. Ultrathin sections were prepared and incubated with Mab-1–12–3 and with protein A-gold complex (15 nm, E-Y Lab., Inc.), followed by staining with aqueous uranyl acetate. Between incubations, the sections were washed with 1% bovine serum albumin phosphate buffer (pH 7.4).

In situ hybridization.
Whole mount in situ hybridization was carried out as described previously (Barth and Wilson, 1995Go; Teraoka et al., 2002Go). Four percent paraformaldehyde (PFA)-fixed embryos were treated with 10 µg/ml proteinase K in PBS with 0.1% Tween 20. After incubation with hybridization buffer containing 50% formamide, 5x SSC, 2 mg/ml Torua RNA and 200 µg/ml heparin (prehybridization) for 1 h, embryos were hybridized with an antisense probe of zebrafish CYP1A, in which the cDNA (483 base pairs [bp]) was originally cloned by degenerate-PCR by K. Yamazaki. Following hybridization overnight at 65°C, embryos were washed with 2x SSC and 0.2 xSSC twice for 30 min, respectively. After blocking with 2% blocking reagent (Roche), embryos were incubated overnight with 4000x diluted anti-DIG antibody conjugated with alkaline phosphatase (Roche) at 4°C. The color reaction was carried out by incubation with BM-purple substrate (Roche).

Statistics.
Results are presented as mean ± SEM. Significance of differences between vehicle and TCDD-exposed groups was determined by 1-way ANOVA, which, if significant, was followed by the Bonferroni/Dunn test (p < 0.05).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TCDD induces local circulation failure in the dorsal midbrain.
Despite reports of circulation failure being consistently observed in TCDD-induced embryos of many fish species (Henry et al., 1997Go; Spitsbergen et al., 1990Go; Walker et al., 1991Go), only subjective assessments of blood flow have been reported. In the present study, local circulation was evaluated by counting the actual number of RBCs passing through a particular vessel with time-lapse recording (Teraoka et al., 2002Go). To quantitate the effect of TCDD exposure on blood flow, the mesencephalic vein (MsV) was selected. It is the only vessel perfusing the dorsal midbrain of the zebrafish embryo at this early stage of development (Fig. 1Go, photo insert).



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FIG. 1. Effects of graded concentrations of waterborne TCDD on blood flow in the mesencephalic vein of the zebrafish embryo from 36 to 96 hpf. Photo inset shows mesencephalic vein (MsV) of the zebrafish embryo. (A) Mesencephalic vein blood-flow results for zebrafish embryos treated with either vehicle (control) or increasing concentrations of TCDD (0.3–1 ppb), beginning at 24 hpf until the time indicated on the abscissa. Blood flow is expressed as the number of red blood cells (RBCs) passing through the mesencephalic vein per 15 s, determined from time-lapse recordings. (B) Mesencephalic vein blood flow evaluated at 2 h intervals from 48 to 60 hpf in vehicle-exposed (control) and TCDD-exposed (0.3 ppb) zebrafish embryos. Results are expressed as mean ± SEM, n = 10. Asterisk indicates a significant difference from control, p <= 0.05.

 
At 36 hpf, circulation was not observed in any vessels of the zebrafish embryo, except around the heart. Vigorous blood flow was first detected in the mesencephalic vein and in other vessels beginning at 46–48 hpf. In control embryos, blood flow was highest in the mesencephalic vein at 50 hpf (Fig. 1AGo) before gradually decreasing to a plateau level around 60 hpf (Fig. 1BGo). This lower plateau level of mesencephalic vein blood flow in control embryos persisted until at least 120 hpf. In TCDD-treated embryos, the RBC perfusion rate in the mesencephalic vein at 50 hpf was similar to control at 0.1 ppb TCDD but was maximally inhibited at 0.3, 0.5, and 1.0 ppb TCDD (Fig. 1AGo). The inhibitory effect on mesencephalic flow appeared to be diminished at 60 hpf because of the decrease in blood flow in the control group at this time. However, a further reduction in perfusion rate was caused by the 3 highest concentrations of TCDD at 72, 84, and 96 hpf, with blood flow almost stopping at the 2 highest TCDD concentrations at 96 hpf (Figs. 1A and 8AGoGo). The magnitude of the inhibitory effect of TCDD on blood flow in the mesencephalic vein was dependent on the TCDD concentration, and at 50 hpf, maximal inhibition was achieved at an apparent concentration of TCDD in the exposure water of 0.3 ppb (Figs. 1A and 8AGoGo).



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FIG. 8. Comparison of TCDD dose-response curves for decreased blood flow and increased apoptosis in the dorsal midbrain of the zebrafish embryo and the inverse correlation between the two responses. (A) The TCDD concentration-response curve for decreased blood flow in the mesencephalic vein at 50 hpf (closed circles) and that for increased apoptosis at 60 hpf (open circles). Dose response curves were constructed from apoptosis results published in Dong et al. (2001) and blood flow results shown in Figure 1Go. (B) The percentage of apoptotic cells in the dorsal midbrain at 60 hpf as a function of the blood flow at 50 hpf in the mesencephalic vein. The graph in B was constructed from results shown in Figures 1, 2, 5, and 6GoGoGoGo and in Dong et al. (2001). Results are expressed as mean ± SEM, n = 10.

 
Effects of AHR modulators on TCDD-induced circulation failure and apoptosis in the dorsal midbrain.
To determine if effects of TCDD on blood flow and apoptosis in the dorsal midbrain are AHR-dependent responses, effects of a different AHR agonist, ß-naphthoflavone (BNF), was studied, and effects of pre-treating TCDD-exposed embryos with an AHR antagonist, {alpha}-naphthoflavone (ANF) were investigated (Dong et al., 2001Go; Rowlands and Gustafsson, 1997Go). With respect to BNF, blood flow in the mesencephalic vein at 50 hpf was inhibited by 1 µM BNF (Fig. 2AGo). Although the results are not shown, this inhibitory effect of BNF on blood flow was concentration-dependent, with 10 nM BNF having no effect and concentrations of 100 nM and 1 µM BNF, causing progressively greater reductions in mesencephalic vein perfusion. Figure 2Go also shows, 10 h later than detection of the decrease in blood flow, that 1 µM BNF caused a significant increase in apoptosis (Fig. 2BGo). This is consistent with the BNF-induced increase in apoptosis in the dorsal midbrain of zebrafish embryos at 60 hpf reported by Dong et al.(2001). Also the BNF concentration-dependence of the 2 responses, reduced blood flow at 50 hpf and increased apoptosis at 60 hpf, are similar in the dorsal midbrain (Dong et al., 2001Go).



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FIG. 2. Effect of exposing zebrafish embryos to the AHR agonist, ß-naphthoflavone (BNF), and TCDD-treated zebrafish embryos to the AHR antagonist, {alpha}-naphthoflavone (ANF), on blood flow and apoptosis in the dorsal midbrain. (A) Blood flow in the mesencephalic vein was determined at 50 hpf. (B) The percent of apoptotic cells in the dorsal midbrain was determined at 60 hpf. Embryos were treated with 1 µM BNF or 500 nM ANF in the presence or absence of 0.3 ppb TCDD. Results are expressed as mean ± SEM, n = 10. Asterisk indicates a significant difference from control, p <= 0.05.

 
With respect to effects of the AHR antagonist ANF, exposure of zebrafish embryos to 500 nM ANF alone had no effect on either blood flow or apoptosis (Figs. 2A and 2BGo, respectively). When exposed to 0.3 ppb TCDD alone, the expected significant decrease in mesencephalic vein perfusion and increase in apoptosis were observed. When zebrafish embryos were co-exposed, beginning at 24 hpf, to both ANF and TCDD, the TCDD-induced decrease in blood flow and the increase in apoptosis were blocked (Figs. 2A and 2BGo, respectively), supporting the notion that both effects are AHR-dependent. Furthermore, the concentration dependency of ANF in blocking the TCDD-induced reduction in mesencephalic vein blood flow was similar to that for blocking the TCDD-induced increase in apoptosis (results not shown). Also, 100 nM ANF was able to prevent 1 µM BNF-induced circulation failure to the dorsal midbrain.

Induction of CYP1A in endothelium of the mesencehpalic vein by TCDD.
It is widely known that TCDD activates the AHR, stimulates transcription of CYP1A mRNA, and induces CYP1A protein in the vascular endothelium of fish and other vertebrates (Stegeman et al., 1995Go). Therefore, the effect of TCDD exposure on CYP1A mRNA transcription in the vascular endothelium of the zebrafish embryo was evaluated by whole-mount in situ hybridization (Fig. 3Go). CYP1A mRNA expression was hardly detected in control embryos at 48 hpf (Fig. 3AGo). However, exposure to a TCDD concentration of 0.1 ppb caused a slight increase in CYP1A message (Fig. 3BGo) and TCDD concentrations ranging from 0.3 to 1.0 ppb evoked striking increases in CYP1A mRNA expression, especially in the head and branchiogenic primordia (Figs. 3C and 3DGo). At these higher TCDD concentrations, the type of tissue in which CYP1A mRNA expression is increased corresponds to the vascular endothelium. This conclusion is also supported by the close similarity between (A) the pattern of increased CYP1A mRNA expression in the head of a 48-hpf zebrafish embryo exposed to 0.5 ppb TCDD (Fig. 3EGo), and (B) a drawing of the blood vessels in the head of a representative zebrafish embryo at the same stage of development by Isogai et al. (2001; Fig. 3FGo). It is noteworthy in the zebrafish embryo that the TCDD concentration dependency for CYP1A induction in the vascular endothelium is similar to that for circulation failure and apoptosis in the dorsal midbrain.



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FIG. 3. Effect of exposure to graded concentrations of waterborne TCDD, beginning at 24 hpf, on expression of CYP1A mRNA in the vascular endothelium of the zebrafish embryo head assessed by whole-mount in situ hybridization at 48 hpf. The representative embryos shown were exposed to either vehicle (A) or 0.1, 0.3, 0.5, or 1.0 ppb TCDD (B, C, E, and D, respectively). Length of the bar in panel E = 100 µm; the same level of magnification was used in A-E. In F, a drawing of blood vessels in the head region of a 48 hpf control zebrafish embryo is kindly provided by Isogai et al. (2001) for comparison to the pattern of CYP1A mRNA expression in E. Abbreviations for the blood vessels identified in E and F are: ACeV, anterior cerebral vein; ACV, anterior cardinal vein; CCV, common cardinal vein; MsV, mesencephalic vein; MtA, metencephalic artery; PMBC, primordial midbrain channel; and PrA, prosencephalic artery.

 
Immunohistochemistry with anti-CYP1A monoclonal antibody (Mab 1–12–3), which is specific for various fish CYP1A proteins (Park et al., 1986Go), was evaluated in 48-hour-old zebrafish embryos exposed to 1 ppb TCDD or vehicle (control) beginning at 24 hpf. It was found that CYP1A protein is induced by TCDD in endothelial cells of blood vessels in the head, including the mesencephalic vein in the dorsal midbrain (Figs. 4A and 4BGo). CYP1A immunoreactivity was not detected in vehicle-exposed control embryos (results not shown). It is significant that induction of CYP1A immunoreactivity in the brain parenchyma, namely in neurons or glia, was never observed, even at higher TCDD concentrations in the exposure water. This localization of increased CYP1A immunoreactivity to vascular endothelial cells was confirmed by immune electron microscopy of blood vessels in the brain (Figs. 4C and 4DGo).



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FIG. 4. Increased expression of CYP1A immunoreactivity in the vascular endothelium in the head of a TCDD-exposed zebrafish embryo at 48 hpf. Embryos were exposed to 1 ppb TCDD beginning at 24 hpf, euthanized at 48 hpf, and fixed for immunohistochemistry (A and B) and immune electron microscopy (C and D). B and D are magnifications of the rectangular area indicated in A and C, respectively. In A, arrows indicate CYP1A immunoreactivity in endothelium of the mesencephalic vein (MsV). In C, arrows point to endothelia and an asterisk designates the lumen of a blood vessel in the dorsal midbrain. The lengths of the bars in A and B represent 50 µm, while in C and D they represent 1 µm.

 
Effects of CYP1A activity inhibitors on TCDD-induced circulation failure and apoptosis in the dorsal midbrain.
Having demonstrated that induction of CYP1A by TCDD was confined to the endothelial cells of blood vessels in the brain, not to neurons or glia, our next objective was to determine the effect of treatment with CYP activity inhibitors on the TCDD-induced decrease in blood flow at 50 hpf and increase in apoptosis at 60 hpf in the dorsal midbrain of the embryo. Two general inhibitors of CYP, SKF525A (SKF, proadifen, 100 µM) and miconazole (Mico, 100 nM) were used. They are different in chemical structure and mode of CYP inhibition. Figure 5Go shows that exposure to SKF alone and Mico alone beginning at 24 hpf had no effect on mesencephalic blood flow at 50 hpf (Fig. 5AGo) or apoptosis in the dorsal midbrain at 60 hpf (Fig. 5BGo). Exposure to TCDD (0.3 ppb) alone caused the expected decrease in mesencephalic blood flow and increase in apoptosis (Figs. 5A and 5BGo, respectively). Significantly, co-exposure to each CYP inhibitor, SKF or Mico, with TCDD blocked the inhibitory effect of TCDD on mesencephalic blood flow (Fig. 5AGo) and the stimulatory effect of TCDD on apoptosis in the dorsal midbrain (Fig. 5BGo). At the concentrations used, SKF was more effective than Mico in inhibiting the effects of TCDD.



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FIG. 5. Inhibitory effects of cytochrome P450 inhibitors on TCDD-induced local circulation failure at 50 hpf (A) and apoptosis at 60 hpf (B) in the dorsal midbrain of the zebrafish embryo. Embryos were exposed to 100 µM SKF-525A (SKF) or 100 nM miconazole (Mico) in the presence or absence of 0.3 ppb TCDD, beginning at 24 hpf. Results are expressed as mean ± SEM, n = 10. Asterisk indicates significantly different from control, p <= 0.05.

 
Effects of antioxidants on TCDD-induced circulation failure and apoptosis in the dorsal midbrain.
Cantrell et al. (1996) reported that TCDD-induced DNA damage and mortality were inhibited by antioxidants in the medaka embryo. Thus, effects of antioxidants on TCDD-induced circulation failure and apoptosis in the dorsal midbrain of the zebrafish embryo were investigated. Two antioxidants were used, N-acetylcysteine (NAC, 50 µM) and ascorbic acid (Asco, 10 mM). Because pH of the ascorbic acid solution was very low, it was adjusted to pH 7.2 with NaOH before exposing zebrafish embryos to it. Figure 6Go shows that exposure to NAC alone or Asco alone, beginning at 24 hpf, had no effect on mesencephalic blood flow at 50 hpf (Fig. 6AGo) or apoptosis in the dorsal midbrain at 60 hpf (Fig. 6BGo). Exposure to TCDD (0.3 ppb) alone caused the expected decrease in mesencephalic blood flow and increase in apoptosis (Fig. 6A and 6BGo, respectively). Co-exposure to each antioxidant, NAC or Asco, with TCDD prevented the inhibitory effect of TCDD on mesencephalic blood flow (Fig. 6AGo) and stimulatory effect of TCDD on apoptosis in the dorsal midbrain (Fig. 6BGo).



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FIG. 6. Effects of exposure to antioxidants on TCDD-induced local circulation failure at 50 hpf (A) and apoptosis at 60 hpf (B) in the dorsal midbrain of the zebrafish embryo. Embryos were treated with 50 µM N-acetylcysteine (NAC) or 10 mM ascorbic acid (Asco) in the presence or absence of 0.3 ppb TCDD, beginning at 24 hpf. Results are expressed as mean ± SEM, n = 10. Asterisk indicates a significant difference from control, p <= 0.05.

 
Effects of caspase inhibitors on TCDD-induced circulation failure and apoptosis in the dorsal midbrain.
It is widely accepted that apoptosis is caused by activation of a series of caspases. Therefore, the effect of exposing zebrafish embryos, beginning at 24 hpf, to the general caspase inhibitor, Z-VAD-FMK (Z-VAD, 300 µM) on TCDD-induced apoptosis and circulatory failure in the dorsal midbrain was assessed. Figure 7Go shows that exposure to Z-VAD alone, beginning at 24 hpf, had no effect on mesencephalic blood flow at 50 hpf (Fig. 7AGo) or apoptosis at 60 hpf in the dorsal midbrain (Fig. 7BGo). Exposure to TCDD (0.3 ppb) alone caused the expected decrease in mesencephalic blood flow and increase in apoptosis (Figs. 7A and 7BGo, respectively). Co-exposure to the general caspase inhibitor, Z-VAD, with TCDD prevented the stimulatory effect of TCDD on apoptosis (Fig. 7BGo) but not the inhibitory effect of TCDD on mesencephalic blood flow in the dorsal midbrain (Fig. 7AGo). Taken together, these results suggest that caspase activation is involved in TCDD-induced apoptosis, but apoptosis is not responsible for the TCDD-induced reduction in mesencephalic vein blood flow.



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FIG. 7. Effects of exposure to a caspase inhibitor on TCDD-induced local circulation failure at 50 hpf (A) and apoptosis at 60 hpf (B) in the dorsal midbrain of the zebrafish embryo. Embryos were exposed to 300 µM Z-VAD-FMK (Z-VAD) in the presence or absence of 0.3 ppb TCDD, beginning at 24 hpf. Results are expressed as mean ± SEM, n = 10. Asterisk indicates a significant difference from control, p <= 0.05.

 
TCDD dose response relationship for local circulation failure and apoptosis in the dorsal midbrain.
Figure 8Go examines the relationship between reduced blood flow in the mesencephalic vein at 50 hpf and increased apoptosis in the dorsal midbrain at 60 hpf in zebrafish embryos exposed to graded concentrations of waterborne TCDD ranging from 0.05 to 1 ppb. Apoptosis results were taken from our earlier study (Dong et al., 2001Go). Figure 8AGo shows that the TCDD dose response curves for the two effects, although in opposite directions, are very similar. More specifically, no significant effect on either blood flow or apoptosis is observed at the lowest concentration of TCDD tested, 0.05 ppb. The lowest concentration of TCDD to produce a significant inhibitory effect on mesencephalic vein blood flow and stimulatory effect on apoptosis is similar, 0.1 ppb. Finally, the maximally effective TCDD concentrations for reduction of blood flow and increase of apoptosis in the dorsal midbrain of the zebrafish embryo are similar: 0.3 and 0.5 ppb, respectively. To further assess the relationship between these two endpoints, the percentage of apoptosis in the dorsal midbrain at 60 hpf was plotted against the RBC perfusion rate in the mesencephalic vein at 50 hpf in zebrafish embryos, under all of these various treatment conditions and at different exposure concentrations of TCDD (Fig. 8BGo). These values were reconstructed from Figures 1, 2, 5, and 6GoGoGoGo and Dong et al. (2001). The main finding was that the occurrence of apoptosis in the dorsal midbrain was inversely correlated with blood flow in the mesencephalic vein (Fig. 8BGo). The correlation coefficient of –0.91 was highly significant.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Blood flow to the dorsal midbrain.
This is the first demonstration of a TCDD-induced reduction in blood flow to a particular brain region, dorsal midbrain, of the zebrafish embryo. A similar effect has not been reported in any other species, so it is a unique finding. RBC perfusion rate in the mesencephalic vein at 50 hpf was almost 2 times greater than that in other vessels of the control zebrafish embryo (Teraoka et al., 2002Go). The reason for the high rate of blood flow is not known, but it may be essential for proper development of neurons and/or glia in the midbrain. Although morphogenesis of the zebrafish heart is almost complete by around 30 hpf, the vascular system of the embryo continues to develop well after this time. Isogai et al.(2001) showed that the mesencephalic vein first emerged at about 48 hpf as the only vessel perfusing the dorsal midbrain. An extensive network of nerve fibers, the tectal neuropile, appeared by 60 hpf, but there were few nerve fibers in the zebrafish dorsal midbrain at 48 hpf (S.W. Wilson and coworkers, personal communication). The reduction in mesencephalic vein blood flow in TCDD-exposed zebrafish embryos in the present study occurred before most other signs of TCDD developmental toxicity were manifested, such as pericardial and yolk sac edema, anemia, reduced lower jaw length, and body growth retardation (Belair et al., 2001Go; Henry et al., 1997Go; Teraoka et al., 2002Go). Furthermore, the reduced blood flow in this midbrain vessel occurred at least 10 h before blood flow is decreased by TCDD in the intersegmental arteries, central artery, optic vein, and hypobranchial artery, demonstrating that it is among the earliest occurring adverse effects of TCDD on the cardiovascular system (Teraoka et al., 2002Go). The decrease in circulation to the dorsal midbrain at 50 hpf is probably a direct effect of TCDD on either development and/or function of the mesencephalic vein. It cannot be secondary to anemia because this is a later occurring effect of TCDD in the zebrafish embryo (Belair et al., 2001Go). Also, it is unlikely that heart failure is involved in causing the local ischemia, because blood flow is not decreased in other vessels at 50 hpf, when the decrease in mesencephalic vein blood flow is detected (Belair et al., 2001Go; Henry et al., 1997Go; Teraoka et al., 2002Go). zfAHR2 and zfARNT2 mRNAs are expressed in the vascular endothelium before and at 50 hpf and CYP1A protein is induced by TCDD in the endothelium at this time (Andreasen et al., 2002bGo). Thus, the transcriptional machinery necessary for TCDD to alter gene expression in the vascular endothelium is available at 50 hpf supporting the notion that TCDD could act directly on the mesencephalic vein endothelium to reduce blood flow. Research with another AHR agonist, PCB 77, also supports this view. PCB 77 has been shown to act directly on porcine aortic endothelial cells in primary culture to induce CYP1A, cause oxidative stress, and increase endothelial cell permeability (Stegeman et al., 1995Go; Toborek et al., 1995Go). Taken together these findings suggest that the endothelium of the mesencephalic vein is a probable site of TCDD action in the zebrafish embryo.

Apoptosis in the midbrain.
An increase in apoptosis in the midbrain and forebrain was discovered in zebrafish embryos exposed to TCDD using the same experimental design as the present study (Dong et al., 2001Go). Although the percentage of apoptosis caused by TCDD was not remarkably high, TCDD increased the rate of apoptosis 3–4 times above the basal level, with high reproducibility. Since apoptosis during normal development has been thought to have physiological significance, the increased apoptosis caused by TCDD might have toxicological relevance. Because there was not a clear border between the forebrain and midbrain, apoptotic cells were counted in the entire mesencephalon. However, we could find only a few apoptotic cells in the forebrain; most were restricted to the dorsal midbrain (Dong et al., 2001Go). Thus, the actual rate of apoptosis in the dorsal midbrain was much higher than the values indicated. In contrast to the reduction in blood flow to the dorsal midbrain, which was maximal at 50 hpf, absent at 60 hpf, and reduced again at 72 and 96 hpf, the increase in apoptosis was delayed (Dong et al., 2001Go). Increased apoptosis in the midbrain and forebrain was first detected at 60 hpf. At this time, the magnitude of the increase in apoptosis was maximal. It was slightly less at 72 hpf and absent at 96 hpf (Dong et al., 2001Go). Thus, in the TCDD-exposed zebrafish embryo, the maximal reduction in blood flow to the midbrain, 50 hpf, preceded the maximal increase in apoptosis, 60 hpf.

Association of decreased blood flow and increased apoptosis in the midbrain.
The time course results raise the possibility of the reduction in blood flow to the dorsal midbrain, contributing to the increase in apoptosis in the midbrain of the zebrafish embryo exposed to TCDD. In the historical experiment that led to the discovery of apoptosis, it was observed in the liver that apoptosis of hepatocytes was caused by occlusion of the portal vein (Kerr et al., 1972Go). Also, in the hippocampus of the gerbil, apoptotic cell death was caused by transient ischemia (Nitatori et al., 1995Go). The mesencephalic vein is the only vessel perfusing the dorsal midbrain of the zebrafish embryo before 96–120 hpf (Isogai et al., 2001Go). This may explain why apoptosis is greatest in this particular brain region following TCDD exposure.

If the decrease in blood flow to the dorsal midbrain contributes to the increase in apoptosis in this brain region, the TCDD dose-response curves for the two effects should be similar, and they should be affected in parallel by treatments that affect TCDD action. Results of the present study and that of Dong et al.(2001) demonstrate that both types of effects are observed. The TCDD dose-response curve for the decrease in blood flow in the mesencephalic vein at 50 hpf is essentially a mirror image of that for the increase in apoptosis at 60 hpf (Fig. 8AGo). Both effects caused by TCDD in the zebrafish embryo showed similar sensitivities to inhibition by an AHR antagonist (ANF), CYP inhibitors (SKF525A and miconazole), and antioxidants (N-acetylcysteine and ascorbic acid). Lastly, under all of these various treatment conditions and at different exposure concentrations of TCDD, the incidence of apoptosis was inversely related to RBC perfusion rate in the mesencephalic vein, with a correlation coefficient of –0.91, supporting the idea that the two responses may be causally related.

AHR dependence.
The local circulation failure and apoptosis in the dorsal midbrain caused by TCDD in the zebrafish embryo was mimicked by exposure to BNF, an AHR agonist, and both effects of TCDD were inhibited by concomitant exposure to ANF, the AHR antagonist (Gasiewicz and Rucci, 1991Go). Based on these results both responses would appear to be AHR-dependent. Two AHR homologs, zfAHR1 and zfAHR2, have been reported in zebrafish and in other fish species (Andreasen et al., 2002aGo,bGo; Hahn, 2001Go; Tanguay et al., 1999Go; Wang et al., 1998Go). However, zfAHR2 appears to be the receptor involved in TCDD toxicity, not zfAHR1 (Andreasen et al., 2002aGo). This is based on the finding in COS-7 cells expressing zfAHR1 and zfARNT2b that TCDD exposure fails to cause significant induction of dioxin-responsive reporter genes. Yet, in identical experiments, TCDD exposure causes significant induction of reporter gene expression in cells expressing zfAHR2 and zfARNT2b (Andreasen et al., 2002bGo). Also zfAHR2, not zfAHR1, exhibits high-affinity binding to radiolabeled TCDD (Andreasen et al., 2002aGo) and mRNA for zfAHR2, not zfAHR1, is expressed in the vascular endothelium of the zebrafish embryo (Andreasen et al., 2002bGo). Taken together, these results suggest that effects of TCDD on the blood flow and apoptosis in the dorsal midbrain of the zebrafish embryo are probably mediated by zfAHR2.

Cytochrome P4501A.
TCDD-induced local circulation failure and apoptosis in the midbrain were inhibited by 2 CYP inhibitors, which are structurally quite different from each other. The TCDD dose-response relationship for CYP1A mRNA expression in the zebrafish embryo appears similar to that for the decrease in blood flow and increase in apoptosis in the dorsal midbrain. As expected, TCDD exposure also increased expression of CYP1A protein, demonstrated immunohistochemically in endothelial cells of various blood vessels in the brain, including the mesencephalic vein. AHR agonist-induction of CYP1A in brain vasculature has long been known (Smolowitz et al., 1991Go), and TCDD-induced expression of CYP1A has also been observed in the brain vasculature of the zebrafish embryo (Andreasen et al., 2002bGo) before and after the time when blood flow in the mesencephalic vein is maximally reduced and apoptosis in the midbrain is maximally increased by TCDD.

Induction of CYP1A in the vasculature of medaka and lake trout larvae exposed to TCDD has been directly related to edema and mortality (Cantrell et al., 1996Go; Guiney et al., 1997Go). Although there are over a hundred different CYP molecules reported, there are no inhibitors that are entirely specific for CYP1A activity. ANF has relative specificity for CYP1A, but it also has AHR antagonistic activity as mentioned above (Tassaneeyakul et al., 1993Go). Considering all of this information, it is possible that TCDD-induced local circulation failure is related to CYP1A induction in the endothelium of the mesencephalic vein. However, CYP1A is also induced in the endothelium of other blood vessels at 50 hpf and blood flow in these vessels is not reduced at this time by TCDD. Thus, the basis for the differential effect of TCDD on blood flow to different regions of the zebrafish embryo when CYP1A is induced throughout the vasculature of the body remains uncertain.

Oxidative stress and vascular permeability.
TCDD induces oxidative stress and tissue damage in the rat brain in vivo, including lipid peroxidation and DNA single-strand breaks (Hassoun et al., 1998Go, 2001Go). Thus, AHR agonists can affect brain function, but the relationship between these events and neurotoxicity remains unclear. TCDD-induced circulation failure and apoptosis in the midbrain of the zebrafish embryo were blocked by two different antioxidants, N-acetylcysteine and ascorbic acid. Cantrell et al.(1996, 1998) also reported that TCDD-induced mortality in medaka embryos was associated with CYP1A induction and was diminished by treatment with N-acetylcysteine. Based on this finding, Cantrell and coworkers proposed that CYP1A induced by TCDD produces oxidative stress that leads to apoptosis in vascular endothelial cells. Dong et al., (2001), using transmission electron microscopy and TUNEL staining failed, however, to detect vascular endothelial cells with apoptotic features in either the brain or other tissues of TCDD-exposed zebrafish embryos at 48 hpf. Also, a transmission electron microscopic assessment of the vascular endothelium of TCDD-exposed lake trout larvae that had elevated levels of CYP1A in the vasculature, and had developed overt signs of developmental toxicity, failed to demonstrate pathological changes in vascular endothelial cells (Guiney et al., 2000Go).

In primary cultures of porcine aorta, endothelial-cell exposure to certain AHR agonists causes CYP1A induction, lipid peroxidation, and an increase in albumin permeability (Stegeman et al., 1995Go; Toborek et al., 1995Go). The increase in albumin permeability is not elicited by PCB congeners that are non-AHR agonists (Toborek et al., 1995Go). PCB 77 produced apoptosis in porcine endothelial cells, but only in the presence of an inhibitor of glutathione production that generates oxidative stress (Slim et al., 2000Go). Although TCDD induces CYP1A in mammals and fish, planar halogenated aromatic hydrocarbons are imperfect substrates for CYP1A, causing uncoupling and release of reactive oxygen species (Schlezinger et al., 1999Go), which could lead to the production of oxidative stress (Alsharif et al., 1994Go). Therefore, AHR activation could increase vascular permeability through mechanisms other than apoptosis of endothelial cells. More specifically, the mesencephalic vein is a fine vessel where only one RBC can pass at a time. It is possible that increased permeability of this vessel could lead to a local decrease in blood flow. Thus, a possible mechanism for future investigation is that TCDD may increase permeability of endothelial cells in the mesencephalic vein secondary to CYP1A induction and oxidative stress, leading to local circulation failure followed by increased apoptosis in the midbrain.

In conclusion, a decrease in blood flow in the mesencephalic vein is one of the earliest-occurring adverse effects of TCDD in the zebrafish embryo. Oxidative stress associated with CYP1A induction in the endothelium of this particular vessel may decrease blood flow to the dorsal midbrain, secondary to an increase in vascular permeability; however, this remains to be demonstrated. Finally, a significant correlation between local circulation failure, and apoptosis in the dorsal midbrain strongly suggests that TCDD induces apoptosis in the midbrain, secondary to the local circulation failure.


    ACKNOWLEDGMENTS
 
This work was supported by grants from the Japanese Ministry of Education, Science, Sports, and Culture, Gakujutsu-Frontier Cooperative Research from active research in Rakuno-gakuen University, Hokkaido Foundation for the Promotion of Scientific and Industrial Technology, Rakuno Scholarship Society, the University of Wisconsin Sea Grant Institute under grants from the National Sea Grant College Program, National Oceanic and Atmospheric Administration, US Department of Commerce, and the National Institutes of Health. Sea Grant Project number R/BT-16 (R.E.P.) and NIH grant ES-07381 (J.J.S.). We deeply thank Dr. S. W. Wilson for providing valuable suggestions on this research.


    NOTES
 
1 To whom correspondence should be addressed. Fax: +81-11–387–5890. E-mail: hteraoka{at}rakuno.ac.jp. Back


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