Columbia Environmental Research Center, U.S. Department of Interior, Geological Survey, 4200 New Haven Rd., Columbia, Missouri 65201
ABSTRACT
The article highlighted in this issue is "2,3,7,8-Tetrachlorodibenzo-p-dioxin Toxicity in the Zebrafish Embryo: Local Circulation 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. 191201).
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 elevated exposure of fish to these chemicals in certain environments. 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 experience 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`s1970`s through a recruitment failure (Cook et al., 1997).
The mechanism of action for dioxin and dioxin-like compounds 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 understood (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 transduction 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.
The overt symptoms of dioxin toxicity in fish embryos and 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, craniofacial deformities, and wasting syndrome (Helder, 1980, 1981
). The cardiovascular system, and in particular the vascular endothelium 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 confirmed 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 endothelium 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 b5 (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 enhance 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 highlighted 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 2426) and vascular dysfunction was seen in the yolk vein (Stage 2829; Cantrell et al., 1996
). Therefore, the present findings are consistent with our observations 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.
NOTES
1 For correspondence via fax: (573) 876-1896. E-mail: donald_tillit{at}usgs.gov.
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