Role of Aryl Hydrocarbon Receptor in Mesencephalic Circulation Failure and Apoptosis in Zebrafish Embryos Exposed to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

Wu Dong*, Hiroki Teraoka*,1, Yoshikazu Tsujimoto*, John J. Stegeman{dagger} and Takeo Hiraga*

* Department of Toxicology, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Japan; and {dagger} Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts

Received August 13, 2003; accepted October 20, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent and potent developmental toxicant in various animals, with developing fish being the most sensitive organisms. Although the expression of aryl hydrocarbon receptor (AHR) as well as the partner molecule, AHR nuclear translocator (ARNT) in the brain has been reported, the effect of TCDD on the brain remains to be clarified in detail. Previously, we reported local circulation failure and apoptosis in dorsal midbrain caused by TCDD in developing zebrafish. In the present experiments, we investigated the effects of morpholino antisense oligos against aryl hydrocarbon receptor 2 (zfAHR2) (AHR2-MO) on toxicological endpoints caused by TCDD in developing zebrafish. AHR2-MO but not its negative homologue (4mis-AHR2-MO) improved TCDD-evoked circulation failure in mesencephalic vein and reduced the occurrence of apoptosis in dorsal midbrain, with concomitant inhibition of CYP1A induction in vascular endothelium. Injection of bovine serum albumin (BSA) into the general circulation, followed by immunohistochemistry with anti-BSA, showed that TCDD raised vascular permeability to albumin in dorsal midbrain, which was blocked by AHR2-MO and N-acetlycystein. In the absence of TCDD, development of embryos injected with AHR2-MO appeared normal at least until 60 h after fertilization. It is concluded that AHR2 activation in the vascular endothelium of the zebrafish embryo midbrain is involved in the mesencephalic circulation failure and apoptosis elicited by TCDD. This is the further evidence that vascular endothelium is the target of TCDD in relation to local circulation failure and apoptosis in dorsal midbrain.

Key Words: apoptosis; aryl hydrocarbon receptor; CYP1A; TCDD; dioxin; oxidative stress.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Early life stages of zebrafish and several other fish species are sensitive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) developmental toxicity (Henry et al., 1997Go; Walker and Peterson, 1994Go). The hallmark endpoints consist of circulatory failure, edema, craniofacial malformation, and growth retardation culminating in mortality (Henry et al., 1997Go; Spitsbergen et al., 1991Go). The cardiovascular system is a key target, and it is also a site of strong induction of cytochrome P4501A (CYP1A) (Spitsbergen et al., 1991Go; Stegeman et al., 1989Go; Tanguay et al., 2003Go), generally accepted as a marker of aryl hydrocarbon receptor (AHR) activation. In mammals, AHR found in the cytoplasm of cells is associated with two molecules of HSP90 and AHR interactive protein (AIP). Upon ligand binding, AHR translocates to the nucleus where it dimerizes with the aryl hydrocarbon receptor nuclear translocators (ARNT). This ligand receptor complex then binds to specific DNA sequences, termed AHR-responsive enhancers (AHREs) that are present in genes, such as CYP1A, whose transcription is regulated by TCDD. Zebrafish AHRs and ARNTs have been cloned and functionally characterized (Andreasen et al., 2002aGo; Tanguay et al., 1999Go, 2000Go, 2003Go). Unlike mammals, there are two forms of the AHR in zebrafish: zfAHR1 and zfAHR2 (Andreasen et al., 2002aGo; Hahn et al., 1997Go). Although the sequence of zfAHR1 is most similar to the mammalian AHR, zfAHR2 causes far greater stimulation of AHREs in response to TCDD. Furthermore, the expression of zfAHR1 is much lower than that of zfAHR2 (Andreasen et al., 2002bGo). Therefore, zfAHR2 is hypothesized to mediate CYP1A induction in zebrafish (Andreasen et al., 2002aGo).

Based on experiments with Ahr-/- mice, it is evident that the toxicity of TCDD is dependent on the AHR (Fernandez-Salguero et al., 1996Go; Mimura et al., 1997Go; Tanguay et al., 2003Go). Several studies in fish embryos point to a possible role for CYP1A and oxidative stress in TCDD developmental toxicity. In the medaka embryo, TCDD causes CYP1A induction and apoptosis in the vitelline vasculature, and both effects are blocked by N-acetylcystein (Cantrell et al., 1996Go, 1998Go). In the zebrafish and killifish embryo brain, blood flow in the mesencephalic vein, the only vessel perfusing the dorsal midbrain early in development, is transiently reduced 50% by TCDD, and this is associated with increased apoptosis in the dorsal midbrain (Dong et al., 2001Go, 2002Go; Toomey et al., 2001Go). This effect of TCDD in zebrafish is also decreased by CYP1A antagonists and by antioxidants (Dong et al., 2002Go).

It is well known that exposure to halogenated aromatic hydrocarbons (HAH) may be associated with long-lasting neurodevelopmental defects in children, as suggested by the poisoning in Yu-Sho and Yu-Cheng (Chen et al., 1992Go), and cognitive deficits in children born in the vicinity of the Great Lakes (Jacobson and Jacobson, 1996Go). Neurological disorders such as sleep disturbances or headache have been observed among some workers who were exposed to TCDD in accidents (Van den Berg et al., 1998Go). In monkeys and rats, profound effects of TCDD and other AHR agonistic PCBs on learning and other ability have been reported by Schantz group (Schantz and Bowman 1989Go; Widholm et al., 2003Go). Many efforts have been made to demonstrate that HAHs reduce long-term potentiation (LTP) in the hippocampus of rats, a possible mechanism effect on memory, especially for ortho-substituted polychrolinated biphenyls (PCBs). On the other hand, expression of AHR, ARNTs, and induction of CYP1A by HAHs have been reported in brain of rats (Huang et al., 2000Go), mice (Shimada et al., 2003Go), and fish (Andreasen et al., 2002bGo; Dong et al., 2002Go; Petersen et al., 2000Go; Powell et al., 2000Go). In relation to this, long-lasting TCDD exposure could induce oxidative stress in rat brain (Hassoun et al., 1998Go). However, the significance of the AHR in the neurotoxic effects by HAH remains unclear.

The present study is focused on the early endpoints of TCDD developmental toxicity expressed in the dorsal midbrain of the zebrafish embryo. It seeks to determine if these endpoints are zfAHR2-dependent. To this end a selective gene knock-down technique using morpholino antisense oligonucleotide (Nasevicius and Ekker, 2000Go) against zfAHR2 (AHR2-MO) was used to decrease the translation of each protein in early stage embryos exposed to vehicle or TCDD. We show that AHR2-MO protected embryos against TCDD-induced increases in vascular permeability, transient decreases in blood flow, and increases in apoptosis in the zebrafish dorsal midbrain. This molecular evidence, together with our separate set of study (Teraoka et al., 2003bGo), establishes that AHR2 is required for these early endpoints of TCDD developmental toxicity to be expressed in zebrafish.


    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). N-Acetylcysteine (NAC) was purchased from Sigma (St Louis, MO). Other chemicals were obtained from Kanto Kagaku (Japan).

Zebrafish embryos and TCDD exposure.
Fertilized eggs were obtained from natural mating of adult zebrafish (AB line) 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. Embryos were collected within 1 h of spawning, rinsed, and placed into a clean Petri dish. Within 24 h post fertilization (hpf) of spawning, the embryos were exposed to either the TCDD vehicle (0.1% DMSO) or to graded, apparent concentrations of waterborne TCDD of 0.3, 0.5, or 1.0 parts per billion (ppb) dissolved in 0.1% DMSO. The DMSO or appropriate concentration of TCDD dissolved in DMSO was present in 3 ml of Zebrafish Ringer 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). Some embryos were exposed with N-acetylcysteine with or without 0.3 ppb TCDD from 24 hpf until 50 hpf.

Gene knock-down with morpholino antisense oligonucleotides.
Based on the cDNA sequence published at Genbank for zfAHR2 (AF063446) (Tanguay et al., 1999Go), a morpholino antisense oligo against zfAHR2 (AHR2-MO) along with a 4 base mismatch negative control morpholino (4mis-AHR2-MO), were synthesized by Gene Tools (Philomath, OR), as described previously (Teraoka et al., 2003bGo). Basic procedures are followed according to Nasevicius and Ekker (2000)Go. Sequences were 5'-TGTACCGATACCCGCCGACATGGTT-3' (AHR2-MO) and 5'-TGaACCcATACCCGCCGtCATcGTT-3' (4mis-AHR2-MO) with four modified bases indicated by lower case letters. Each morpholino was injected into the yolk of embryos at the one- or two-cell stage with a fine glass needle connected to an automatic injector (IM-300, Narishige, Japan). The volume of the 100 µM morpholino solution injected into each embryos was 100 pl. Occasionally, injected embryos died before 10 hpf, depending on the state of the tip of the glass needle and embryos. Thus 5 or 10 surviving embryos were used for each experiment.

Blood flow in the dorsal midbrain.
Blood flow in the mesencephalic vein was evaluated by time-lapse recording using a digital-video camera (Teraoka et al., 2002Go). Embryos (50 hpf) were suspended in 200 µl of 3% carboxymethyl cellulose/Zebrafish Ringer solution in a plastic bath mounted on the stage of an inverted microscope (IMT-2, Olympus, Japan). Temperature of the suspension solution was maintained at 28.5°C with a PDMI-2 Micro-Incubator with Bipolar Temperature Controller (TC-202, Medical Systems, Greenvale, NY).

Conventional histology.
At 60 h post fertilization (hpf), embryos were fixed in 10% neutralized formalin for 24 h, followed by the conventional procedure for TUNEL staining (Dong et al., 2001Go, 2002Go). Positive TUNEL signals were detected with ABC KIT (Elite, Vector), and brain sections were counterstained with methyl green. TUNEL-positive cells in both forebrain and midbrain were counted in all serial sections of these brain regions in each embryo, because there was no clear morphological border between the dorsal midbrain and forebrain (Dong et al., 2001Go). Immunohistochemistry was performed using a monoclonal antibody specific for fish CYP1A (Mab 1-12-3) (Dong et al., 2002Go). Signals were detected with ABC Kit (Vector), with brown color distinct from black pigment in the skin, the eye, and the midline. Methyl green was used for counterstaining.

Vascular permeability.
To assess vascular permeability in the embryonic midbrain in vivo, immunohistochemistry using anti-bovine serum albumin (BSA) was performed. At 50 hpf embryos were injected with 10% BSA into the sinus venosus and fixed with 4% paraformaldehyde after 10 min of BSA injection. The same protocol described above for antibody staining was used, except that the primary antibody was an anti-BSA rabbit antibody (Sigma, St Louis, MO) (153 ng/ml), and the secondary antibody was an anti-rabbit IgG goat antibody (Sigma, St Louis, MO) (7.5 µg/ml). For quantification of vascular permeability, area analysis of BSA immunoreactivity was performed using conventional image software (Photoshop 7.0; Adobe). After several images of the sagital section of dorsal midbrain with vasculature were obtained with digital camera (Penguin 150CL, Pixera) under light microscope (BX50, Olympus), the anti-BSA-immonoreactive area was circled on the Photoshop image at the same magnification, and the area was obtained in pixels.

Statistics.
Results are presented as mean ± SE. Significant differences between means were determined by one-way ANOVA followed by Scheffe’s test (p < 0.05).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of AHR2-MO on the TCDD-Induced Transient Decrease in Mesencephalic Blood Flow
The only blood vessel perfusing the dorsal midbrain (optic tectum) of control zebrafish embryos around 48 hpf is the mesencephalic vein. In control embryos, blood flow in this vessel reaches a peak around 50 hpf and then gradually decreases to a basal level by 58 hpf (Fig. 1AGo). In TCDD-exposed embryos (0.3 ppb), the transient increase in mesencephalic blood flow, seen in the control group from 48 to 56 hpf, was essentially abolished (Fig. 1AGo). However, embryos injected with the AHR2-MO before they were exposed to this same concentration of TCDD (0.3 ppb), exhibited none of the inhibitory effects on blood flow (Fig. 1AGo). Figure 1BGo shows results for mesencephalic blood flow for various treatment groups at 50 hpf. Mesencephalic vein blood flow in embryos exposed to vehicle only (control), vehicle + AHR2-MO, and vehicle + 4mis-AHR2-MO were similar. In contrast, embryos exposed to graded concentrations of TCDD (0.3, 0.5, and 1 ppb) displayed markedly lower blood flow that appeared to be dose related at the lowest TCDD concentrations used. The major effect of the AHR2-MO in embryos exposed to TCDD was that it blocked the inhibitory effect on mesencephalic vein blood flow caused by the lowest concentration of TCDD (0.3 ppb) but not by the two higher concentrations (0.5 and 1 ppb). When the negative control AHR2 morpholino (4mis-AHR2-MO) was injected in embryos exposed to 0.3 ppb TCDD, the inhibitory effect of TCDD on blood flow was still observed, demonstrating selectivity of the AHR2-MO.



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FIG. 1. Effect of treatment with AHR2-MO on TCDD-induced decreases in mesencephalic vein blood flow in the zebrafish embryo. (A) Uninjected, vehicle-exposed embryos (Control: filled circles), uninjected TCDD-exposed embryos (TCDD: open circles), or AHR2-MO-injected and TCDD-exposed embryos (AHR2-MO + TCDD: open triangles) were exposed continuously to an apparent waterborne concentration of 0.3 ppb TCDD beginning at 24 hpf until observation at 48–58 hpf. Height of each bar and associated vertical line is mean ± SEM (n = 7–10 embryos/treatment). (B) After receiving either no morpholino injection, or an injection of either AHR2-MO (or MO) or its negative homolog (4mis-AHR2-MO or 4mis), embryos were exposed to vehicle or graded concentrations of TCDD (0.3, 0.5, or 1 ppb). At 50 hpf the number of red blood cells perfusing the mesencephalic vein per 15 s was used as an index of blood flow. Height of each bar and associated vertical line is mean ± SEM (n = 10 embryos/treatment). The presence of an asterisk (*) indicates a significant difference from respective control group. Level of significance was set at p <= 0.05.

 
Effect of AHR2-MO on TCDD-Induced Increase in Apoptosis in the Dorsal Midbrain
TCDD produces a small but significant increase in the incidence of apoptosis in the forebrain and dorsal midbrain of zebrafish larvae at 60–72 hpf, which is correlated with the decrease in circulation in the mesencephalic vein at 50 hpf (Dong et al., 2002Go). TUNEL-positive signals in the midbrain showed round or lunar shape and did not accompany erythrocytes (Fig. 2Go insets; Dong et al., 2001Go). To determine if this endpoint is AHR2 dependent, the effect of AHR2-MO pretreatment on TCDD-induced apoptosis in these brain regions was investigated (Fig. 2Go). The percentage of apoptotic cells in vehicle (control), vehicle + AHR2-MO, and vehicle + 4mis-AHR2-MO treated embryos was similar, 0.1–0.2%. Exposure to graded concentrations of TCDD resulted in dose-related increases in apoptosis to 0.4–0.5%. As observed for blood flow, the AHR2-MO only blocked the TCDD-induced increase in apoptosis at the lowest TCDD concentration used (0.3 ppb). At the two higher concentrations (0.5 and 1 ppb TCDD) the AHR2-MO was without effect. Also at the lowest TCDD concentration, the negative control zfAHR2 morpholino, 4mis-AHR2-MO, was ineffective in blocking the TCDD-induced increase in apoptosis.



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FIG. 2. Effect of the AHR2-MO on TCDD-induced increases in apoptosis in the dorsal midbrain of the zebrafish embryo. Uninjected embryos and embryos injected with either AHR2-MO or 4mis-AHR2-MO were exposed from 24 to 60 hpf to one of three graded concentrations of TCDD (0.3, 0.5, or 1.0 ppb). At 60 hpf the embryos were fixed for TUNEL staining. Results are expressed as percentage of apoptotic cells (TUNEL positive) in complete serial sections of the forebrain and midbrain. Height of each bar and associated vertical line is mean ± SEM (n = 10 embryos/treatment). All other conditions are the same as described in the legend to Figure 1Go. Inset images show the representative images of TUNEL-positive cells in dorsal midbrain (arrow heads). There are ten TUNEL-positive cells between two arrow heads (six round nuclei and four condensed chromatin between two arrows). Right image is the magnification of the left (square). Bar = 50 µm.

 
Effect of AHR2-MO on the TCDD-Induced Increase in CYP1A Expression
In TCDD-exposed embryos, zebrafish cytochrome P4501A (zfCYP1A) induction in the midbrain was restricted to the vascular endothelium (Dong et al., 2002Go). There was only minimal CYP1A immunostaining in vehicle-exposed (control) embryos at 48 hpf (Fig. 3AGo), in contrast to the marked increase in CYP1A expression in TCDD-exposed embryos (Figs. 3CGo and 3EGo). TCDD (0.3 ppb) markedly induced CYP1A expression not only in the endothelium of the brain vasculature and in other blood vessels throughout the body but also in the skin and the pharyngeal arches (Fig. 3C and EGo). In vehicle-exposed embryos treated with the AHR2-MO, there was no obvious effect on the pattern or degree of CYP1A immunostaining observed (Fig. 3BGo). However, in embryos exposed to the lowest concentration of TCDD (0.3 ppb), pretreatment with the AHR2-MO essentially blocked the increase in CYP1A immunostaining in both the brain vasculature and pharyngeal arches (Fig. 3DGo). However, CYP1A induction caused by higher concentrations of TCDD (0.5 and 1 ppb) was not prevented by AHR2-MO injection in mesencephalic endothelial cells, while CYP1A induction in the skin and branchogenic primordial was effectively blocked (Fig. 3E and FGo). This observation was reminiscent of the effects of AHR2-MO on circulation failure and apoptosis. The 4mis-AHR2-MO did not show any effect on CYP1A immunostaining in the representative vehicle-exposed embryo (Fig. 3GGo), nor did it alter either the pattern or intensity of CYP1A immunostaining in TCDD-exposed embryos (Fig. 3HGo).



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FIG. 3. Effect of treatment with a morpholino against zfAHR2 on immunostaining for zfCYP1A in a lateral, longitudinal section of the head of a representative control or TCDD-exposed zebrafish embryo. Results are shown for a representative embryo in eight different treatment groups. Each embryo was exposed to 0.1% DMSO (vehicle control), 0.3 ppb TCDD, or 0.5 ppb TCDD for 24–48 hpf. Immunostaining for zfCYP1A was conducted on all embryos at 48 hpf. Representative embryos in the following treatment groups are shown: uninjected embryos exposed to vehicle (A), 0.3 ppb TCDD (C), and 0.5 ppb TCDD (E); embryos injected with AHR2-MO and exposed to vehicle (B), 0.3 ppb TCDD (D), and 0.5 ppb TCDD (F); embryos injected with the 4 base mismatch AHR2-MO (4mis: negative control) and exposed to vehicle (G) or 0.3 ppb TCDD (H). Arrows, arrowheads, and asterisks indicate CYP1A immunoreactivity in the pharyngeal arches, in the cranial vascular endothelium, and in the skin, respectively. Double arrows in (A) and (F) express black pigment in the skin or the midline. Bar = 100 µm.

 
TCDD-Induced Increase in Mesencephalic Vein Permeability to Albumin and the Effect of AHR2-MO
To evaluate vascular permeability, a bovine serum albumin (BSA) solution was injected into the sinus venosus of zebrafish embryos at 50 hpf. Embryos were then fixed for immunohistochemistry with anti-BSA antibody (Fig. 4Go). In vehicle-exposed embryos, BSA immunoreactivity was restricted to the lumen of the blood-vessel-like structure in the dorsal midbrain (Fig. 4A and BGo). On the other hand, in a representative TCDD-exposed embryo, BSA immunoreactivity was observed between cells in the brain parenchyma (arrowheads), suggesting that vascular permeability to albumin was increased by TCDD in this brain region (Fig. 4C and DGo). In TCDD-exposed embryos, AHR2-MO blocked the scattering of BSA immunoreactivity in the brain parenchyma (Fig. 4E and FGo). In TCDD-exposed embryos treated with the negative control 4mis-AHR2-MO, the apparent increase in mesencephalic vein permeability to albumin was not prevented, demonstrating specificity of the AHR2-MO (results not shown). Furthermore, treatment of zebrafish embryos with N-acetylcysteine (50 µM), which has been shown to block the decrease in mesencephalic vein blood flow and the increase in apoptosis caused by TCDD (Dong et al., 2002Go), blocked the invasion of BSA immunoreactivity into the dorsal midbrain parenchyma of TCDD-exposed embryos (Fig. 4G and HGo).



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FIG. 4. Effect of treatment with a morpholino against zfAHR2 on immunostaining for bovine serum albumin (BSA) in a section of the dorsal midbrain of a representative control or TCDD-exposed zebrafish embryo. Results are shown for a representative embryo (n = 5) in four different treatment groups. Each embryo was exposed to either 0.1% DMSO (vehicle control) or 0.3 ppb TCDD from 24–50 hpf. At 50 hpf embryos were injected with a BSA solution into the sinus venosus and fixed for immunostaining with anti-BSA antibody 10 min after the injection. Representative embryos in the following treatment groups are shown: uninjected embryos exposed to either vehicle (A and B) or TCDD (C and D); embryos injected with AHR2-MO and exposed to TCDD (E and F); and embryos treated with N-acetylcysteine (NAC) and exposed to TCDD (G and H). Arrowheads in D indicate scattering of BSA immunoreactivity in parenchyma. All right panels (B, D, F and H) are magnified photos of respective left panels (A, C, E, and G). Arrows and arrowheads indicate BSA immunoreactivity within cranial vessels and between brain mesenchymal cells, respectively. Nonspecific signals were recognized in the skin (*). Bars = 50 µm. Representative images of five embryos.

 
Using area analysis of BSA immunoreactivity outside vascular lumen with conventional imaging software, vascular permeability was quantified and expressed in pixels (Fig. 5Go). As shown in Fig. 5Go, 0.3 ppb TCDD caused significant increase of albumin permeability in dorsal midbrain, and this effect was almost abolished by AHR2-MO and NAC treatment.



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FIG. 5. Quantitative representation of the effect of a morpholino against zfAHR2 on the vascular permeability to bovine serum albumin in dorsal midbrain. The vascular albumin permeability was quantified with image analysis of sagittal sections of BSA immunostaining and was expressed in pixels. Experimental protocol was the same as Fig. 4Go. Height of each bar and associated vertical line is mean ± SEM (n = 5 embryos/treatment).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study provides the first molecular evidence for involvement of zfAHR2 in dorsal midbrain endpoints of TCDD developmental toxicity in zebrafish. The endpoints were all manifested early in development, 50–60 hpf, and included decreased blood flow and increased permeability in the mesencephalic vein and increased apoptosis in the dorsal midbrain. These findings are consistent with those of an earlier study where the AHR agonist (ß-naphthoflavone) and antagonist ({alpha}-naphthoflavone) were used to implicate AHR in the circulation failure and apoptosis in the dorsal midbrain of the zebrafish embryo (Dong et al., 2001Go).

Since zfAHR1 and zfAHR2 have little sequence similarity near their ATG start sites, it was expected that AHR2-MO would decrease zfAHR2 protein levels selectively without altering levels of zfAHR1. Consistent with this expectation, addition of AHR2-MO to an in vitro transcription and translation reaction containing zfAhr1 and zfAhr2 cDNAs blocked translation of zfAHR2 with no effect on zfAHR1 (Prasch et al., 2003Go). Furthermore, TCDD induction of zfCYP1A, an AHR-dependent gene, was reduced by pretreatment with AHR2-MO, demonstrating that this effect of TCDD in the zebrafish embryo is mediated by zfAHR2.

The increased bovine serum albumin (BSA) immunostaining in the dorsal midbrain area of TCDD-exposed embryos provided direct evidence of a TCDD-induced increase in vascular permeability. Since the increased BSA immunostaining of the midbrain parenchyma was reduced in zfAHR2 exposed to TCDD, it suggests that zfAHR2 is involved in mediating this response to TCDD. In addition, pretreatment with an antioxidant also blocked the increase in vascular permeability caused by TCDD in the dorsal midbrain, which is entirely consistent with our earlier finding that TCDD-induced circulation failure and apoptosis in the zebrafish dorsal midbrain is markedly reduced by antioxidant treatment (Dong et al., 2002Go). In medaka embryos treated with TCDD, general circulation failure and mortality also were blocked by antioxidant (Cantrell et al., 1996Go). Although we reported a close relationship between circulation failure in the mesencephalic vein, the only vessel in dorsal midbrain at this stage, and apoptosis in the dorsal midbrain in the previous report (Dong et al., 2002Go), it is still not certain whether apoptosis could be caused by reduction in blood flow by about half. On the other hand, it is also possible that unidentified substance inducing apoptosis could be released from endothelium or leaked out from blood constituents (Teraoka et al., 2003aGo).

It appears that the vascular endothelium of the zebrafish embryo brain, where zfCYP1A is strongly expressed, is the site of toxic action involving CYP1A (Dong et al., 2002Go). Endothelium has been suggested as a possible site of AHR agonist toxicity ever since high levels of CYP1A were identified in endothelium (Stegeman et al., 1989Go). In porcine aortic endothelial cell culture, the AHR agonist PCB 77 increases both lipid peroxidation and albumin permeability, and the latter is reduced by antioxidant (Slim et al., 2000Go; Toborek et al., 1995Go). Furthermore, the increase in albumin permeability is specific for AHR agonists and is usually accompanied by CYP1A induction in the endothelial cells (Toborek et al., 1995Go). The results here support the idea that CYP1A in the vasculature may be involved in a variety of toxic effects of TCDD and other planar halogenated aromatic hydrocarbons. Both AHR2-MO and an antioxidant prevented TCDD-induced mesencephalic circulation failure and apoptosis in the dorsal midbrain of the zebrafish embryo. It is possible in TCDD-exposed zebrafish embryos that oxidative stress is caused by TCDD uncoupling of CYP1A resulting in the production of activated oxygen, as shown with a planar polychlorinated biphenyl AHR agonist (Schlezinger et al., 1999Go). Recent studies have shown that TCDD also stimulates ROS release from induced scup liver microsomes (Goldstone and Stegeman, unpublished), consistent with this suggestion. However, induced levels of CYP1A could lead to radical formation originating from increased rates of some endogenous compound metabolism, and this also could be involved in eliciting oxidative stress. While the mechanism by which CYP1A may be involved in causing apoptosis in the zebrafish embryo midbrain is not known, the ability of an antioxidant to block the effect suggests that oxidative stress in the vascular endothelium of this brain region may be involved.

Unlike Ahr -/- null mice that exhibit cardiac hypertrophy, alterations in vascular maturation, and disrupted immune and female reproductive function including impaired ovarian follicle growth (Abbott et al., 1999Go; Benedict et al., 2000Go; Fernandez-Salguero et al., 1996Go; Lahvis et al., 2000Go), no effects of the AHR2-MO were detected on gross morphology or vascular development of zebrafish embryos at or before 60 hpf. Therefore, even though zfAHR2 and zfARNT2 were expressed by 12 hpf (Andreasen et al., 2002bGo), the physiological function of zfAHR2 in early stage embryos is yet to be determined. In contrast, injection of zebrafish embryos with a negative splice variant form of zfARNT2, zfARNT2X, caused severe defects in brain, eye, pectoral fin, heart, and gut development (Hsu et al., 2001Go).

In contrast to the observations at the lower concentration of TCDD, AHR2-MO was without effects on mesencephalic circulation failure and apoptosis at higher concentration of TCDD (0.5 and 1 ppb). Similarly, AHR2-MO did not affect CYP1A induction caused by higher concentration of TCDD in mesencephalic vascular endothelium. Reminiscent of these observations, {alpha}-naphthoflavone and antioxidants blocked mesencephalic circulation failure and apoptosis by 0.3 ppb TCDD but not by 0.5 and 1 ppb TCDD (Dong et al., 2001Go, 2002Go). Thus, these facts raise the possibility that different mechanisms do exist in TCDD-induced toxicity dependent on the concentration of TCDD. However, we do not know whether our AHR2-MO treatment blocked AHR2 translation completely, since specific antibody to AHR2 is not available at present. As our experiments were performed with 48, 50, or 60 hpf embryos, the effectiveness of the molpholino was expected to decrease. AHR2-MO completely lost its preventive activity against TCDD by 96 hpf, when pericardial edema, general circulation failure, and death occurred (Teraoka et al., 2003bGo). Thus, we cannot exclude the possibility that the residual AHR2 could mediate toxicity by higher concentrations of TCDD. Although higher concentrations of TCDD could cause some effects via pathways other than those engaged at lower concentrations of TCDD, 0.3 ppb TCDD caused almost maximal effects in mesencephalic circulation failure and apoptosis (Dong et al., 2001Go, 2002Go). Interestingly, the magnitude of AHR2-MO effect on CYP1A induction caused by TCDD was different in different regions. Thus, AHR2-MO effectively inhibited CYP1A induction even by 0.5 and 1 ppb TCDD in the skin or pharyngeal arches; the effect of morpholino was slight for CYP1A induction in cranial vascular endothelium at those doses of TCDD. Although CYP1A expressed in the skin might be involved in TCDD-induced toxicity as a barrier to ambient water (Andreasen et al., 2002bGo), present endpoints were not correlated to CYP1A induction in the skin, as revealed by different effects of AHR2-MO. Further investigations are required to clarify these issues.

In conclusion, AHR2-MO prevented TCDD-induced mesencephalic circulation failure and apoptosis in the dorsal midbrain of the zebrafish embryo. This suggests the conserved role of AHR in TCDD-induced toxicity for all vertebrates. Mesencephalic vein seems a target of TCDD for the production of these effects (Dong et al., 2002Go). The mechanism by which TCDD causes apoptosis in the zebrafish embryo midbrain is still not known, but the ability of an antioxidant to block the effect suggests that oxidative stress in the vascular endothelium of this brain region may be involved.


    ACKNOWLEDGMENTS
 
This work was supported by grants-in-aid for scientific research and grant-in-aid for JSPS fellows from the Japanese Ministry of Education, Culture, Sports Science, and Technology (MEXT) (T.H. H.T., and W.D.), cooperative research from Rakuno Gakuen University Gakujutsu-Frontier (T.H. and H.T.), cooperative research from active research in Rakuno Gakuen University 2002–8 (H.T.), and EPA grant R827102-01-0 and NIH grant ES07381 (J.J.S.).


    NOTES
 
1 To whom correspondence should be addressed at Department of Toxicology, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu 069-8501, Japan. Fax: +81-11-387-5890. E-mail: hteraoka{at}rakuno.ac.jp. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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