* Department of Toxicology, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu 069-8501, Japan;
Department of Veterinary Anatomy, University of Tottori, Japan;
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
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ABSTRACT |
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Key Words: apoptosis; CYP1A; circulation failure; cytochrome P4501A; developmental toxicity; TCDD; dioxin; endothelial cell; midbrain; zebrafish embryo; aryl hydrocarbon receptor; oxidative stress; mesencephalic vein.
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INTRODUCTION |
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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., 1989). It was found that TCDD-induced mortality was preceded by circulatory failure in larvae of various fish species (Elonen et al., 1998
; Henry et al., 1997
; Spitsbergen et al., 1990
; Walker and Peterson, 1994
). 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., 1997
). 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., 1999
). 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., 1996
, 1998
). Finally, results in TCDD-exposed zebrafish and lake trout larvae also suggest that vascular endothelial function is disrupted (Guiney et al., 2000
; Henry et al., 1997
).
It is notable that these findings are consistent with the association between TCDD exposure and cardiovascular disease in humans (Calvert et al., 1998; Flesch-Janys et al., 1995
), a role for the aryl hydrocarbon receptor (AHR) in vascular remodeling during fetal development in mice (Lahvis et al., 2000
), 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., 1995
; Toborek et al., 1995
), 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., 2000
).
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., 1990). TCDD also causes injury to rat hippocampal neurons in culture, which is associated with an increase in intracellular calcium (Hanneman et al., 1996
). We discovered that TCDD induces apoptotic cell death in the dorsal midbrain of early zebrafish embryos (Dong et al., 2001
) and a similar observation has been reported in Fundulus heteroclitus embryos following TCDD exposure (Toomey et al., 2001
). In zebrafish embryos, the effect of TCDD was mimicked by the AHR agonist, ß-naphthoflavone (BNF), and was inhibited by the AHR antagonist,
-naphthoflavone (ANF), suggesting that TCDD-induced apoptosis in the dorsal midbrain of zebrafish larvae is an AHR-dependent response (Dong et al., 2001
).
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., 2002b). 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.
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MATERIALS AND METHODS |
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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, 1995). 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 Ringers 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., 2001; Gavrieli et al., 1992
). 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., 2001
).
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., 2002). 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 1123, Park et al., 1986). 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-1123) (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., 1991
). 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-1123 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, 1995; Teraoka et al., 2002
). 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).
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RESULTS |
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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., 1995). 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. 3
). CYP1A mRNA expression was hardly detected in control embryos at 48 hpf (Fig. 3A
). However, exposure to a TCDD concentration of 0.1 ppb caused a slight increase in CYP1A message (Fig. 3B
) 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 3D
). 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. 3E
), 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. 3F
). 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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DISCUSSION |
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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., 2001). Although the percentage of apoptosis caused by TCDD was not remarkably high, TCDD increased the rate of apoptosis 34 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., 2001
). 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., 2001
). 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., 2001
). 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., 1972). Also, in the hippocampus of the gerbil, apoptotic cell death was caused by transient ischemia (Nitatori et al., 1995
). The mesencephalic vein is the only vessel perfusing the dorsal midbrain of the zebrafish embryo before 96120 hpf (Isogai et al., 2001
). 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. 8A). 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, 1991). 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., 2002a
,b
; Hahn, 2001
; Tanguay et al., 1999
; Wang et al., 1998
). However, zfAHR2 appears to be the receptor involved in TCDD toxicity, not zfAHR1 (Andreasen et al., 2002a
). 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., 2002b
). Also zfAHR2, not zfAHR1, exhibits high-affinity binding to radiolabeled TCDD (Andreasen et al., 2002a
) and mRNA for zfAHR2, not zfAHR1, is expressed in the vascular endothelium of the zebrafish embryo (Andreasen et al., 2002b
). 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., 1991), and TCDD-induced expression of CYP1A has also been observed in the brain vasculature of the zebrafish embryo (Andreasen et al., 2002b
) 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., 1996; Guiney et al., 1997
). 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., 1993
). 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., 1998, 2001
). 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., 2000
).
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., 1995; Toborek et al., 1995
). The increase in albumin permeability is not elicited by PCB congeners that are non-AHR agonists (Toborek et al., 1995
). 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., 2000
). 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., 1999
), which could lead to the production of oxidative stress (Alsharif et al., 1994
). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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