* School of Pharmacy and the Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin 53705
1 To whom correspondence should be addressed at the School of Pharmacy, University of Wisconsin, 777 Highland Ave., Madison, WI 53705-2222. Fax: (608) 265-3316. E-mail: wheidema{at}wisc.edu.
Received October 9, 2003; accepted December 4, 2003
ABSTRACT
A common response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in teleost embryos is blue-sac disease, characterized by pericardial and yolk-sac edema. The cellular and extracellular fluids of freshwater fish are hyperosmotic compared to the surrounding water. In order to be in osmotic balance, freshwater fish must maintain a barrier to minimize water entry and excrete excess water that passes the barrier. We hypothesized that edema observed in TCDD-exposed zebrafish was caused by a failure of a barrier to incoming water. As a test of this hypothesis, we removed the osmotic gradient that drives water entry by increasing the osmolarity of the surrounding water with mannitol. Abolishing the osmotic gradient between the interior body fluids and the water environment of the developing zebrafish significantly reduced both pericardial and yolk-sac edema. When added after edema formation had already started, mannitol only partially reversed pre-existing edema. An alternate hypothesis is that TCDD impairs water excretion, allowing water to accumulate as edema fluid. However, we were unable to demonstrate an alteration in kidney function: expression of early markers for kidney development appeared normal, and we did not observe TCDD-induced changes in kidney filtration. An alteration in the overall shape of the kidney was observed, but this may be a consequence of compression by edema. In conclusion, TCDD exposure may inhibit the function of a permeability barrier to water, which is critical for maintaining osmotic balance in early development.
Key Words: AHR2; ARNT2; TCDD; zebrafish; blue-sac syndrome; edema.
Polychlorinated dibenzo-p-dioxins (PCDDs) are persistent, lipophilic, bioaccumulative pollutants (Safe, 1990). The most potent PCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes several toxic responses in developing teleost fish mediated through the aryl hydrocarbon receptor (AHR2) pathway (Peterson et al., 1993
; Prasch et al., 2003
; Tanguay et al., 2003
; Walker and Peterson, 1994
). One such response is blue-sac syndrome, a noninfectious disease characterized, particularly, by pericardial and yolk-sac edema, which can be induced after exposure to many chemicals. One well-documented example where the occurrence of blue sac syndrome has been linked to exposure to TCDD and structurally related compounds (via maternal transfer) is in laboratory-reared larvae of wild lake trout from Lake Ontario (Cook et al., 2003
). Other signs of TCDD toxicity include arrested growth, craniofacial malformations, cardiac malformation, anemia, and mortality (Elonen et al., 1998
; Henry et al., 1997
).
The zebrafish is an excellent model system for studying the effects of TCDD on developing teleost fish (Andreasen et al., 2002; Belair et al., 2001
; Dong et al., 2002
; Henry et al., 1997
; Tanguay et al., 2003
). Zebrafish exposed to TCDD immediately after fertilization develop edema as early as 72 h postfertilization (hpf) (Henry et al., 1997
). Interestingly, if exposure to TCDD is delayed until after 96 hpf, edema is not observed (Belair et al., 2001
). This suggests that developing zebrafish are especially vulnerable to TCDD shortly after hatching.
Because freshwater fish are constantly surrounded by a medium of very low osmolarity, the ability to exclude water is essential. Freshwater fish are dependent on gills, kidney, and skin to maintain proper internal volume and osmolarity. The skin forms a barrier to water entry, while water that gets past this barrier is constantly being excreted by the kidneys. Cardiovascular function is essential for kidney development (Serluca et al., 2002) as well as gill and kidney functions.
During the first two days of development, the embryo must maintain osmotic balance without the aid of the kidney. The chorion is not essential for this process, as dechorionated embryos can exclude water. This indicates the existence of a water permeability barrier on the surface of the developing embryo. This barrier may consist of two separate barriers: one surrounding the embryo and one surrounding the yolk (Hagedorn et al., 1998). Between 48 and 72 hpf, development of the gills and digestive system may produce areas of increased permeability (Kimmel et al., 1995
). An attractive possibility is that TCDD disrupts the maintenance of a barrier against the inward diffusion of water. This model is consistent with a previous report in which blood vessel permeability was increased by TCDD exposure in lake trout larvae (Guiney et al., 2000
).
A second potential model is that edema results from decreased water export. This could result from direct effects of TCDD on kidney development or function, or from an indirect effect in which decreased blood circulation inhibits glomerular formation and filtration. Alternatively, effects of TCDD on the pronephric ducts might affect electrolyte and solute reabsorption, possibly disturbing the osmotic balance within the fish.
The teleost pronephros consists of a pair of nephrons with two glomeruli fused at the midline in a region ventral to the aorta. Paired bilateral ducts are connected to the glomerulus via pronephric tubules (Drummond et al., 1998; Tytler, 1988
; Tytler et al., 1996
). Key genes involved in regulating nephrogenesis include pax 2a (Krauss et al., 1991
), sim 1 (zebrafish single-minded homolog), and the zebrafish wt 1 homolog (Serluca and Fishman, 2001
). In the zebrafish, filtration of blood derived from the dorsal aorta begins in the glomerulus at approximately 48 hpf (Drummond et al., 1998
; Majumdar and Drummond, 1999
). Filtrate passes through fenestrations in the capillary endothelium into Bowman's space and through the tubules into the pronephric ducts (Saxén, 1987
). While the gills also play a role in osmoregulation, TCDD is unlikely to cause blue sac syndrome in zebrafish via effects on the gills, because the gills do not function in an osmoregulatory capacity until 7 days postfertilization (Rombough, 2002
), well beyond the onset of TCDD-induced edema.
If TCDD disrupts a water permeability barrier, then elimination of the osmotic force that drives water into the fish would decrease water influx and prevent TCDD-induced edema. In this study this was investigated by using mannitol to raise the osmolarity of the water surrounding the developing zebrafish. The morphology and function of the developing pronephros was also examined to determine whether the mechanisms involved in eliminating water from the body were also affected. Our results suggest that TCDD exposure may disrupt the formation or maintenance of a barrier-to-water influx that is necessary for proper osmotic management. Although TCDD consistently produced an alteration in the overall shape of the glomerulus, possibly associated with internal pressure caused by progressive edema formation, markers for nephron development and pronephric function early in development failed to indicate any significant TCDD-induced effect on water elimination from the body. We propose a model in which chemical insults such as those produced by TCDD, which causes edema in fish early-life stages, leads to progressive failure of the renal and circulatory systems, culminating in an irreversible spiral of edema and organ failure that ends in mortality.
MATERIALS AND METHODS
Zebrafish lines and embryos.
AB-line zebrafish were used for all experiments, and were raised and maintained as previously described (Westerfield, 1995) with 50% water changes daily. When appropriate, fish were anesthetized with tricaine (MS222; Sigma) at 1.67 mg/ml.
Waterborne TCDD exposure of embryos.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) of >99% purity (from Chemsyn, Lenexa, KS), was dissolved in 0.1% DMSO. Newly fertilized eggs, approximately 3-4 h (hpf), were exposed to vehicle (0.1% DMSO) or to 0.1, 0.5, 1, 5, or 10 ng/ml TCDD for 1 h in glass scintillation vials with gentle rocking. Following the 1-h static exposure, the embryos were rinsed with water and maintained in vehicle/TCDD-free water for the remainder of the experiments.
Mannitol exposure.
To determine whether TCDD disrupts a water permeability barrier, the osmotic force that drives water into the fish was removed through the addition of mannitol. TCDD- and control-treatment groups, exposed as previously described, were divided so that half of each group was maintained continuously in either normal aquarium water (1 mOsm/l) or in 250 mM mannitol (
250 mOsm/l), respectively. In experiments designed to test the ability of mannitol to reduce pre-existing edema, the fish were treated as described (placed after TCDD exposure into either water or 250 mM mannitol) and then five embryos were removed from each group at 96, 120, and 144 hpf, photographed, and moved into one well of a 24-well plate containing the opposite water type (water vs. 250 mM mannitol). Embryos were not switched before 96 hpf to allow for sufficient edema fluid to collect, so that any decrease in pericardial or yolk-sac area could be reliably detected. For embryos switched at 96 hpf, images were taken of each embryo at 1, 7, 27, and 50 h post change. For embryos switched at 120 hpf, images were taken at 1, 3, 5, 7, and 27 h post change. For embryos switched at 144 hpf, images were taken at 1, 3, 5, and 7 h post change. The time course of TCDD-induced mortality prevented extended observations of the fish switched into mannitol at 144 hpf. All observations were made blind regarding water type and TCDD treatment. Mannitol concentrations within a range between 250 mM and 175 mM concentrations effectively reduced edema formation (data not shown). As concentrations in excess of 175 mM tended to remove internal water content and compress the tissues in early embryos, 175 mM mannitol was used in studies that examined kidney shape.
Edema.
Edema incidence was scored as the percent of live embryos showing any discernable signs of edema at the indicated time (Henry et al., 1997). The extent of edema in individual zebrafish was estimated using digital photographs, in which the cross-sectional area of yolk sac and pericardium were determined using the area measurement function of Scion Image. All pictures were taken at x4 with the fish in a lateral orientation at the same resolution. Area measures are reported as the number of pixels within the outlined area. Observations suggested that part of the edema measurement in TCDD-mannitol-exposed larvae was due to failure to reabsorb the yolk. Therefore, in the water change experiments shown in Figure 3, images were taken with the remaining yolk in focus. Yolk-sac area measurements in these experiments were carried out as described above, and then the area of the remaining yolk was subtracted from the total yolk-sac area to give the final yolk-sac edema value.
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Whole-mount in situ hybridization was used to examine whether the expression patterns of key genes involved in regulating nephrogenesis were affected by exposure to 1 ng/ and 5 ng/ml TCDD. This was performed essentially as described (Oxtoby and Jowett, 1993), using probes to pax 2a (Krauss et al., 1991
), sim 1 (zebrafish single-minded homolog), and the zebrafish wt 1 homolog (Serluca and Fishman, 2001
). These probes labeled the pronephric duct and tubule, pronephric duct, and glomerular primordia, respectively (Drummond et al., 1998
; Serluca and Fishman, 2001
). All riboprobes were labeled with digoxigenin (DIG; in accordance with Roche DIG RNA labelling protocol) and detected using anti-DIG alkaline phosphatase (AP) (1:4000, vol:vol) and BCIP/NBT substrate. Control embryos that were not incubated in riboprobe were also exposed to the substrate to examine possible background staining. Stained embryos were embedded in JB-4 resin as previously described and sectioned transversely at 6 µm.
Endogenous AP activity in the pronephric ducts of embryos exposed to DMSO, 1 and 5 ng/ml TCDD was detected by incubating 4-µm JB-4 tissue sections in AP substrate (0.34 mg/ml NBT, 0.18 mg/ml BCIP, 100 mM Tris pH 9.5, 100 mM sodium chloride, 50 mM magnesium chloride) directly onto the slides for 4560 min. Sections were rinsed in PBST (1% PBS with 0.1% Tween-20) before imaging.
Pronephros Na+/K+ ATPase was detected, as previously described by Drummond et al. (1998), using the monoclonal antibody alpha 6F (Developmental Studies Hybridoma Bank) raised against the chicken -1 subunit (Takeyasu et al., 1988
). This antibody cross-reacts with the zebrafish form of the enzyme (Drummond et al., 1998
). Specific binding was detected with a goat antimouse IgG-HRP secondary antibody (Santa Cruz Biotechnology). The signal was dependent on the primary antibody and did not appear in controls, in which the primary antibody was omitted. Embryos exposed as described for in situ hybridization were fixed in ethanol:DMSO (80:20) prior to whole-mount immunocytochemistry performed as described (Dent et al., 1989
). Labelled embryos were embedded in JB-4 resin and sectioned transversely at 4-µm.
Morpholinos.
The zfahr2 morpholino (MO) sequence (Gene Tools, Corvallis, OR) is complementary to zebrafish AHR2 cDNA (GenBankTM accession #AF063446). The zfahr2-MO (5'-GTA CCG ATA CCC TCC TAC ATG GTT-3') overlapped the translation start site of zfahr2 from 4 bp upstream to 18 bp downstream of the AUG start codon. The Gene Tools standard control morpholino (5'-CTC TTA CCT CAG TTA CAA TTT ATA-3') was used as a control (control-MO). Morpholinos were diluted to 0.1 mM in 1X Danieau's solution (58 mM NaCL, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5 mM HEPES, pH 7.6) as described (Nasevicius and Ekker, 2000).
To determine whether TCDD-induced edema or effects on the kidney were zfAHR2-dependent, embryos were injected with 15 ng of either the zfahr2-MO or control-MO at the 1-2 cell stage as described (Nasevicius and Ekker, 2000
) and allowed to develop for approximately 2 h, after which damaged and unfertilized embryos were discarded. As the zfahr2-MO was fluorescein-tagged at the 3' end, injection success was assessed by fluorescence microscopy. Embryos were then exposed as previously described to vehicle control or TCDD, assessed for edema, and prepared for glomerular histology as described above.
Glomerular function.
As described previously (Drummond et al., 1998), filtration of the blood by the glomeruli was investigated using a qualitative assay. Solutions of lysine-fixable rhodamine green dextran (10,000 molecular weight [mw]) or fluorescein dextran (3000 mw; from Molecular Probes, 1% in PBS) were injected into the sinus venosus using a glass micropipette. Perfusion of the circulatory system was examined by fluorescence microscopy at approximately 5-10 min post injection, before embryos were fixed and sectioned as previously described. Images of the glomeruli from embryos exposed to DMSO, 1 ng and 5 ng/ml TCDD, were taken using a Nikon eclipse TE300 microscope.
Statistics.
Two-way ANOVA or ANOVA and post hoc comparison tests (Tukey or Dunnetts) were performed. Levene's test for homogeneity of variances was performed before the ANOVA. Data that did not pass Levene's test (experiments generating all-or-none responses that are not normally distributed) were subjected to the Mann-Whitney U test. All statistical analysis was performed using the Statistica 6.0 software package. Results are presented as mean ± SE; level of significance was p 0.05.
RESULTS
Osmotic Support Blocks Edema
TCDD-treated embryos clearly gain an excess of water as development progresses, leading to the characteristic edema of blue sac syndrome (Fig. 1). One possible cause for this is increased water influx due to failure of a water permeability barrier. If this is the case, then at least part of the edema should be prevented by the addition of an osmotically active compound to the water surrounding the fish, obviating the force driving water inwards. To be effective, the molecular weight of this compound must be sufficiently large to be excluded from transport into the zebrafish. Mannitol was chosen for these experiments because it is a commonly used osmolyte that does not readily cross between biological compartments. As can be seen in Figure 1, increasing the osmolarity of the water surrounding the developing zebrafish with 250 mM mannitol produced a significant reduction in both pericardial and yolk-sac edema.
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Further experiments were conducted to determine whether increased osmolarity could reverse edema after it had formed. In these experiments, embryos were treated with TCDD and cultured in water, where they showed progressive increases in edema. They were then transferred into 250 mM mannitol at 96, 120, or 144 hpf to determine if mannitol would reverse the edema already formed at these time points. The fish were periodically monitored for reduction in edema severity until 168 hpf. Addition of mannitol at 96, 120, and 144 hpf arrested all further increase in pericardial edema and at 120 and 144 hpf was able to partially reverse the TCDD-induced pericardial edema (Fig. 3C, p < 0.05). In contrast, transfer into mannitol at all times slowed or stopped the development of yolk-sac edema but did not result in any decrease in the yolk-sac area (Fig. 3D).
TCDD and Nephrogenesis
The edema caused by TCDD could also stem from inhibition of kidney formation or function. In such a model, TCDD would cause edema by impairing water excretion by the kidney. Therefore, we investigated the development and function of the pronephros in zebrafish exposed to TCDD.
The sim 1, pax 2a, and wt 1 genes are essential regulators of nephrogenesis in zebrafish and higher vertebrates (Drummond et al., 1998; Kreidberg et al., 1993
; Serluca and Fishman, 2001
; Torres et al., 1995
). TCDD exposure at concentrations of 1 and 5 ng/ml failed to alter the expression pattern of these genes during nephrogenesis. sim 1, an early marker for the developing pronephric duct, was evident from the 2-somite stage in the intermediate mesoderm as bilateral stripes extending caudally from the fourth somite (Fig. 4A and 4B). Expression was later evident in the ventrolateral portion of the somite until 30 hpf when sim 1 expression was no longer present in the pronephros. Expression of the pax 2 zebrafish ortholog pax 2a was observed at 24 hpf, primarily in the anterior portion of the pronephric duct (Fig. 4C and 4D) and by 33 hpf was observed in precursor cells of the pronephric tubule anterior and medial to the duct. The patterns described here match those previously documented (Drummond et al., 1998
; Krauss et al., 1991
; Pfeffer et al., 1998
; Püschel et al., 1992
). The nephron primordia were identified by in situ hybridization with a wt 1 probe (Fig. 4E and 4F). By 33 hpf, paired primordia expressing wt1 had migrated medially to the midline and later fused at 50 hpf, forming the glomerulus beneath the notochord and aorta (Fig. 4G and 4H). TCDD did not appear to have an effect on the expression pattern of these important markers for nephrogenesis, nor did TCDD block migration and midline fusion. This suggests that TCDD produces no major impairment in early kidney development.
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An injection of fluorescently labeled dextran into the bloodstream allowed visualization of kidney filtration of fluid into the tubules (Drummond et al., 1998). Dextran was filtered through the glomerulus into the tubules and duct lumen in both TCDD- and DMSO-treated embryos (Fig. 5I and 5K). The dextran also revealed normal development of the capillary beds in the glomeruli of both control and treated fish.
Although the pronephros of TCDD-exposed fish appeared to show the normal developmental pattern of marker expression and signs of function at 56 hpf, in transverse section, the glomeruli of TCDD-exposed fish were consistently flattened in appearance as shown in Figure 6B. Although the glomerular area remained unchanged in exposed larvae, the medial height of each glomerulus was reduced, and the width was increased in a TCDD dose-related manner (Fig. 6I). In addition to the alteration in shape, TCDD tended to produce a reduction in the area of Bowman's space, as though increasing pressure from edema formation might be flattening the glomerulus. This change in shape induced by TCDD was largely reversed when fish were grown in mannitol, in which the pericardial and yolk-sac edemas were reduced (Fig. 6D). Despite the presence of TCDD, zebrafish reared in 175 mM mannitol developed a compact spherical glomerulus, lacking the flattened appearance normally seen in TCDD-exposed fish. This suggests that the flattened appearance of the glomerulus is due to compression from edema.
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DISCUSSION
Maintaining body osmolarity is a fundamental problem faced by freshwater fish. In order to survive, fish must maintain a barrier against water influx and continually export water that passes the barrier. The distinctive edema in blue-sac syndrome caused by TCDD and other AHR agonists indicates a defect in either the water barrier and/or the water-export system.
Water Barrier
In mammals, TCDD has well documented adverse effects on the skin, including most notably the production of chloracne in humans and certain mammalian species (Kimmig and Schulz, 1957; Martinez et al., 2003
). The skin and fins of juvenile fish exposed to TCDD are also adversely affected by TCDD. Fin necrosis, cutaneous hyperpigmentation, and hemorrhage occur in juvenile fish exposed to acutely toxic doses of TCDD (Walker and Peterson, 1994
). In TCDD-exposed zebrafish embryos and larvae, AHR activation occurs in the skin and is reflected by an increase in cytochrome P4501A expression (Andreasen et al., 2002
). Cutaneous hemorrhage is also observed in the skin of embryos and larvae of zebrafish and other fish species exposed to TCDD (Tanguay et al., 2003
). However, the dermal toxicity of TCDD in fish is not as well understood as in mammals. These effects on the primary barrier between the organism and its environment may extend to early development, prior to the completion of skin formation. In such a model, TCDD increases surface permeability to water at a critical period in early zebrafish development, resulting in the formation of edema that is a hallmark sign of blue-sac syndrome.
The existence of a water permeability barrier at the surface of the zebrafish embryo can be inferred from the fact that zebrafish embryos are able to maintain osmotic balance prior to the development of organs such as the pronephros that can export water. Disruption of this barrier by TCDD could explain the manifestation of edema characteristic in exposed zebrafish larvae. Using mannitol to increase the osmolarity of the water surrounding the developing zebrafish successfully blocked the edema formation induced by TCDD exposure. This therefore demonstrated that an effect on a permeability barrier was a likely contributing factor to this sign of TCDD toxicity.
Rescuing edematous zebrafish by transferring the fish to mannitol after edema had formed was less successful. Presumably, as the effect of mannitol is to remove the osmotic gradient that drives water into the fish, the most likely explanation for this result is that there is no strong driving force for removing fluid from the yolk sac and pericardium once these compartments have become expanded. The fluid volume in these compartments might be reduced by hyperosmotic surroundings, but fluid volume in other compartments such as the circulatory system are likely to be reduced as well by such a treatment.
Water Export
Water export in freshwater fish is dependent on proper kidney and circulatory function. Prior studies have shown that TCDD impairs circulatory function beginning between 60 and 72 hpf, depending on the vessel examined (Dong et al., 2002; Henry et al., 1997
). This is close to the time when edema is first detectable. However, examination of the documented phenotypes of zebrafish with mutations that affect cardiac function (including heartstrings, heart of glass, leaky heart, pickwick, gridlock, beach bum, jekyll, and slow motion) where circulation slows or stops, suggests that TCDD-induced circulatory failure is not solely responsible for edema formation. These fish primarily develop pericardial edema (also starting around 72 hpf) with little or no yolk-sac edema (Alexander et al., 1998
; Chen et al., 1996
; Sprague et al., 2001
; Warren et al., 2000
). Thus circulatory failure does not adequately explain the severe yolk-sac edema seen in TCDD-exposed zebrafish larvae.
TCDD might also produce defects in kidney development and/or function. In this case, mannitol would also have had an effect on edema formation by slowing the rate of water influx to compensate for a reduced rate of water export. However, little evidence was found to indicate that TCDD disrupts pronephric development. For control and TCDD-treated embryos, the expression of the key pronephric developmental genes sim 1, pax 2a, and wt 1 appeared to follow the patterns previously described (Drummond et al., 1998; Kreidberg et al., 1993
; Serluca and Fishman, 2001
; Torres et al., 1995
).
Exposure to TCDD caused no obvious gross morphological changes to the ducts or tubules, and the overall size of the glomerulus remained normal. The in-growth of capillaries into the glomerulus was apparent in TCDD-exposed embryos, and at 56 hpf, fluorescently labelled dextran filtrate had passed into the duct lumen, suggesting normal filtration. Expression of the Na+/K+ ATPase membrane-bound ion pumps in the pronephric ducts and normal membrane polarity were maintained in the TCDD-exposed embryos. These results tend to rule out a role for dioxin-induced malfunction of the pronephros in causing edema in the early stages of development. Comparison to phenotypes of zebrafish with mutations affecting kidney formation or function (bazooka joe, big league chew, blowup, bubblicious, cyster, dizzy gillespie, double bubble, elipsa, fleer, hubba bubba, inflated, junior, lawrence welk, and pao pao tang) further support this conclusion. These fish develop primarily pericardial edema without the severe balloon-like yolk-sac edema seen in TCDD-exposed zebrafish larvae (Drummond et al., 1998; Sprague et al., 2001
). Overall, these results support a model in which TCDD exposure in some way increases water permeability across the surface of the developing zebrafish.
However, a change in the overall shape of the glomerulus in fish exposed to TCDD was consistently observed. The fact that this flattening of the glomerulus was blocked by mannitol suggests that the change in morphology may be a secondary effect, in which edema pressure distorts the shape of internal organs. Likewise, as previously reported, zfahr2-MO prevented edema formation (Prasch et al., 2003) and also blocked glomerular flattening, thereby demonstrating that both these effects are dependent on AHR2 expression.
Nephrotic syndrome, in which plasma protein alterations lead to movement of fluid from the blood stream into tissues, is also a source of edema (Kaysen 1994). Hypo-osmolarity of the serum, leading to increased fluid transfer from the blood to the tissues, might be an internal cause of the observed edema; however, there is currently no evidence in fish to support this theory. Likewise, increased vascular permeability previously reported in TCDD-exposed larvae in early life stages (Guiney et al., 2000) might also contribute to these blue-sac disease symptoms.
Despite the possibility that the change in kidney morphology is secondary to edema resulting from an effect of TCDD on water permeability, this change in kidney morphology may provide important insight into the cause of a long-recognized phenomenon in which many different kinds of stress can lead to irreversible blue-sac syndrome. The flattened kidney suggests that progressive edema would, at some point, cause decreased kidney function, which in turn would worsen edema. Increased edema might also produce negative effects on the circulatory system, again leading to decreased glomerular filtration and increased edema. Thus, a model (Fig. 7) emerges in which the observed negative impacts on the water permeability barrier, future effects on kidney function, or impaired circulation can each trigger a positive feedback loop affecting the other processes, and producing steadily worsening edema. This model might explain the long-recognized fact that many different kinds of insults can lead to the same universally fatal edematous state in early-life-stage fish. Once activated, these positive feedback loops produce a pathological state that is irreversible.
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ACKNOWLEDGMENTS
The alpha-6F antibody developed by D. M. Fambrough was obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. This work was supported by the University of Wisconsin Sea Grant Institute under grants from the National Sea Grant College Program, National Oceanic and Atmospheric Administration, U.S. Department of Commerce, and Sea Grant Project Numbers R/BT-16 and R/BT-17 (W.H. and R.E.P.). Contribution 354, Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, WI 53726-4087.
NOTES
2 These authors contributed equally to this article.
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