TOXICOLOGICAL SCIENCES 69, 1–2 (2002)

Copyright © 2002 by the Society of Toxicology

TOXICOLOGICAL HIGHLIGHT
2,3,7,8-Tetrachlorodibenzo-p-dioxin Toxicity in the Zebrafish Embryo:
Local Circulation Failure in the Dorsal Midbrain
Is Associated with Increased Apoptosis
Donald E. Tillitt 1 and Diana M. Papoulias
Columbia Environmental Research Center, U.S. Department of Interior, Geological Survey, 4200 New Haven Rd., Columbia, Missouri 65201

The overt symptoms of dioxin toxicity in fish embryos and

The article highlighted in this issue is “2,3,7,8-Tetrachlorod-
ibenzo-p-dioxin Toxicity in the Zebrafish Embryo: Local Circula-
tion Failure in the Dorsal Midbrain Is Associated with Increased
Apoptosis” by W. Dong, H. Teraoka, K. Yamazaki, S. Imani, T.
Imagawa, J. J. Stegeman, R. E. Peterson, and T. Hiraga (pp.
191–201).
Dioxins and dioxin-like compounds have threatened and in
some cases continue to threaten populations of fish. This is due
to an inherent sensitivity of fish during development and ele-
vated exposure of fish to these chemicals in certain environ-
ments. Fish embryos and fry are known to be sensitive to the
developmental effects of dioxin. The early developmental
stages of all vertebrates are susceptible to the untoward effects
of dioxin, but the developing embryos of oviparous vertebrates
are particularly prone to the adverse effects of dioxins. This
fact combined with an elevated exposure risk that fish experi-
ence through water, sediment, and dietary sources of dioxin has
led to reduced and, in some cases, failed recruitment of young
fish into breeding populations. For example, populations of
lake trout in the Great Lakes were thought to be limited by
dioxin-like contaminants (PCBs, PCDDs, and PCDFs) in the
1960⬘s–1970⬘s through a recruitment failure (Cook et al.,
1997).
The mechanism of action for dioxin and dioxin-like com-
pounds is generally accepted to function through the aryl
hydrocarbon receptor (AhR). The pathway from binding of
dioxin to the AhR and subsequent transcriptional upregulation
of the AhR gene battery is fairly well established and under-
stood (Nebert et al., 2000). The genes known to be regulated
through the AhR include drug-metabolizing enzymes, of which
cytochrome P450 or CYP1A is the hallmark. This signal trans-
duction pathway from AhR binding to gene expression in fish
has been better understood in recent years. However, the role
of these genes in dioxin-induced toxicity is still not understood.
1
For correspondence via fax: (573) 876-1896. E-mail: donald_tillitt@
usgs.gov.
1
fry have been well documented. Symptoms of dioxin poisoning
in fish embryos and fry include edema of the yolk sac and
pericardium, hemorrhaging in the head and tail regions, cranio-
facial deformities, and wasting syndrome (Helder, 1980, 1981).
The cardiovascular system, and in particular the vascular en-
dothelium of the developing embryo has been identified as a
primary target of dioxin-induced toxicity (Stegeman et al.,
1989; Guiney et al., 1997). Yet a variety of other tissues,
including those of the gill and digestive tract, are also known
target tissues for AhR agonists (Smolowitz et al., 1991). The
myriad of effects and tissues affected by dioxins has made it
difficult to delineate the primary effects caused by dioxin from
the secondary events that might occur from a general cascade
of effects toward eventual toxicity. The paper by Dong et al.
(2002) is an important step in understanding the sequence of
events that lead to the later symptoms of toxicity in larval fish.
The highlighted article by Dong et al. (2002) is an elegant
combination of physiological level measures with mechanistic
aspects of pharmacology and toxicology. The authors con-
firmed previous findings of dioxin-induced apoptosis in the
brain (Cantrell et al., 1996; Toomey et al., 2001; Dong et al.,
2001), which was reduced through co-treatment with inhibitors
of cytochrome P450. The authors made whole organism level
measurements of blood flow in the mesencephalic vein that
supplies tissues of the dorsal midbrain during this stage of
development. These measurements are a truly unique aspect of
the research and this paper. The technique for quantitative
measurement of blood flow in the microvasculature of the
developing embryo that was developed by the authors (Teraoka
et al., 2002) was key to the understanding developed from this
paper. The authors were able to determine that blood flow in
the mesencephalic vein of the zebrafish was quantitatively
reduced and that this reduction occurred prior and in inverse
proportion to apoptosis in the dorsal midbrain. Another key
finding was that a caspase inhibitor abolished apoptosis in the
dorsal midbrain of the zebrafish without a reduction in blood
flow in the mesencephalic vein. The authors conclude that
circulatory failure and oxidative stress in the vascular endo-
2 TOXICOLOGICAL HIGHLIGHT
thelium are primary events in dioxin-induced toxicity, while
the apoptosis in the neural cells of the dorsal midbrain is a
secondary effect.
Oxidative stress, first identified as an important component
of dioxin-induced toxicity in mammals (Stohs et al., 1983) is
now thought to be a critical event in the pathway(s) toward
dioxin-induced toxicity (Nebert et al., 2000). CYP1A can be a
source of reactive oxygen species through inefficient coupling
of the P450 complex with NADPH-P450 oxidoreductase or
cytochrome b 5 (Schlezinger et al., 1999). Moreover, induction
of CYP1A and oxidative stress in endothelial cells leads to an
increase in membrane permeability (Stegeman et al., 1995).
Ischemia and circulatory failure, which could lead to or en-
hance localized oxidative stress, has also been demonstrated in
a number of fish species exposed to dioxin (Spitsbergen et al.,
1990; Walker and Peterson, 1994; Elonen, 1998). However, the
placement of these events in a logical or sequential model of
dioxin-induced toxicity was not previously evident. The high-
lighted studies of Dong et al. (2002) have clearly helped to
understand the sequence of events leading to loss of neural
cells via apoptotic processes in the dorsal midbrain. Dioxin-
induced apoptosis in the digestive tract, gills, neural tissues,
and endothelial cells of the vascular system were present in
Medaka treated with dioxin (Cantrell et al., 1996, 1998). Our
hypothesis was that oxidative stress leads to apoptosis in the
vascular endothelium and subsequently to loss of vascular
integrity (Cantrell et al., 1996, 1998). In these studies, we only
found apoptosis of the neural tissues in late stage embryos
(Stage 33) of the Medaka, well after apoptosis was observed in
the medial yolk vein (Stage 24 –26) and vascular dysfunction
was seen in the yolk vein (Stage 28 –29; Cantrell et al., 1996).
Therefore, the present findings are consistent with our obser-
vations in Medaka. Clearly, circulatory disruption was prior to
the loss of neural cells to apoptosis. An inconsistency between
our studies is the observation of apoptosis in the endothelial
cells. The differences may be due to species-specific responses.
In either case, the new findings of Dong et al. (2002) provide
clear evidence for the sequence of events in apoptotic loss of
neural cells in the dorsal midbrain. It will be most interesting
to understand the functional changes that may occur as a result
of those losses. Any functional changes in neurobehavioral
responses induced by dioxin will provide a closer linkage to
understanding potential effects on populations.
REFERENCES
Cantrell, S. M., Lutz, L. H., Tillitt, D. E., and Hannink, M. (1996). Embryo-
toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): The embryonic
vasculature is a physiological target for TCDD-induced DNA damage and
apoptotic cell death in Medaka (Orizias latipes). Toxicol. Appl. Pharmacol.
141, 23–34.
Cantrell, S. M., Joy-Schlezinger, J., Stegeman, J. J., Tillitt, D. E., and Hannink,
M. (1998). Correlation of 2,3,7,8-tetrachlorodibenzo-p-dioxin–induced ap-
optotic cell death in the embryonic vasculature with embryotoxicity. Toxi-
col. Appl. Pharmacol. 148, 24 –34.
Cook, P. M., Zabel, E. W., and Peterson, R. E. (1997). The TCDD toxicity
equivalence approach for characterization of trout early life stage mortality
risks associated with exposures to TCDD and related chemicals. In Chem-
ically-Induced Alterations in the Functional Development and Reporduction
of Fishes. (R. M. Rolland, M. Gilbertson, and R. E. Peterson, Eds.), pp.
9 –27. SETAC Technical Publication Series.
Dong, W., Teraoka, H., Kondo, S., and Hiraga, T. (2001). 2, 3, 7, 8-Tetra-
chlorodibenzo-p-dioxin induces apoptosis in the dorsal midbrain of ze-
brafish embryos by activation of aryl hydrocarbon receptor. Neurosci. Lett.
303, 169 –72.
Elonen, G. E., Spehar, R. L., Holcombe, G. W., Johnson, R. D., Fernandez, J. D.,
Erickson, R. J., Tietge, J. E., and Cook, P. M. (1998). Comparative toxicity of
2,3,7,8-tetrachlorodibenzo-p-dioxin to seven freshwater fish species during
early life-stage development. Environ. Toxicol. Chem. 17, 472– 483.
Guiney, P. D., Smolowitz, R. M., Peterson, R. E., and Stegeman, J. J. (1997.)
Correlation of 2,3,7,8-tetrachlorodibenzo-p-dioxin induction of cytochrome
P4501A in vascular endothelium with toxicity in early life stages of lake
trout. Toxicol. Appl. Pharmacol. 143, 256 –273.
Helder, T. (1980). Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on
early life stages of the pike (Esox lucius L.). Sci. Total Enviro. 14, 255.
Helder, T. (1981). Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on
early life stages of rainbow trout (Salmo gairdneri, Richardson). Toxicology
19, 101–112.
Nebert, D. W., Roe, A. L., Dieter, M. Z., Solis, W. A., Yang, Y., and Dalton,
T. P. (2000). Role of the aromatic hydrocarbon receptor and [Ah] gene
battery in the oxidative stress response, cell cycle control, and apoptosis.
Biochem. Pharmacol. 59, 65– 85.
Schlezinger, J. J., White, R. D., and Stegeman, J. J. (1999). Oxidative inacti-
vation of cytochrome P-450 1A (CYP1A) stimulated by 3,3⬘,4,4⬘-tetrachlo-
robiphenyl: Production of reactive oxygen by vertebrate CYP1As. Mol.
Pharmacol. 56, 588 –597.
Smolowitz, R. M., Hahn, M. E., and Stegeman, J. J. (1991). Immunohisto-
chemical localization of cytochrome P450IA1 induced by 3,3⬘,4,4⬘-tetra-
chlorobiphenyl and 2,3,7,8-tetrachlorodibenzoafuran in liver and extrahe-
patic tissues of the teleost Stenotomus chrysops (scup). Drug Metab. Dispos.
19, 113–123.
Spitsbergen, J. M., Walker, M. K., Olson, J. R., and Peterson, R. E. (1990).
Pathologic alterations in early life stages of lake trout, Salvelinus namay-
cush, exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin as fertilized eggs.
Aquat. Toxicol. 19, 41–72.
Stegeman, J. J., Miller, M. R. and Hinton, D. E. (1989). Cytochrome P450IA1
induction and localization in endothelium of vertebrate (teleost) heart. Mol
Pharmacol 36, 723–729.
Stegeman, J. J., Hahn, M. E., Weisbrod, R., Woodin, B. R., Joy, J. S., Najibi, S.,
and Cohen, R. A. (1995). Induction of cytochrome P4501A1 by aryl hydrocar-
bon receptor agonists in porcine aorta endothelial cells in culture and cyto-
chrome P4501A1 activity in intact cells. Mol. Pharmacol. 47, 296 –306.
Stohs, S. J., Hassan, M. Q., and Murray, W. J. (1983). Lipid peroxidation as a
possible cause of TCDD toxicity. Biochem. Biophys. Res. Commun. 111,
854 – 859.
Toomey, B. H., Bello, S., Hahn, M. E., Cantrell, S., Wright, P., Tillitt, D. E.,
and Di Giulio, R. T. (2001). 2,3,7,8-Tetrachlorodibenzo-p-dioxin induces
apoptotic cell death and cytochrome P4501A expression in developing
Fundulus heteroclitus embryos. Aquat. Toxicol. 53, 127–138.
Teraoka, H., Dong, W., Ogawa, S., Tsukiyama, S., Okuhara, Y., Niiyama, M.,
Ueno, N., Peterson, R. E., and Hiraga, T. (2002). 2,3,7,8-Tetrachlorod-
ibenzo-p-dioxin toxicity in the zebrafish embryo: Altered regional blood
flow and impaired lower jaw development. Toxicol. Sci. 65, 192–199.
Walker, M. K., and Peterson, R. E. 1994. Toxicity of 2,3,7,8-tetra-
chlorodibenzo-p-dioxin to brook trout (Salvelinus fontinalis) during early
development. Environ. Toxicol. Chem. 13, 817– 820.