Zebrafish embryo monitoring of the aquatic environment: dose-response synergism revealed in combinations of pollutant chemical mixtures.
In our experiments, embryos were collected each morning, sorted, and staged. At gastrulation [5.25 h post fertilization (hpf)] the embryos were placed for 20 s in a 1.6 [micro]g/ml solution of trypsin (Sigma), made up in embryo rearing solution (ERS) (Carolina Biological); the embryos were then rinsed three times in ERS. This treatment softened the chorion sufficiently that it could be removed with a pair of forceps. The dechorionated embryos were placed in a petri dish containing ERS, and lined with Parafilm (to prevent sticking). A 250-[micro]l droplet of the various chemical pollutants was placed in a petri dish (100 mm) lined with Parafilm, and a single embryo was placed in each droplet. After 30 min of exposure, the embryos were removed from the droplets, rinsed three times in ERS, and transferred into Parafilm-lined petri dishes half-filled with ERS. The embryos were examined immediately after removal from the pollutant, every 24 h until they reached the swim-up stage (96 hpf). Deviations from normal development were video recorded. When the swim-up stage was reached, a final video recording was made and the embryos were killed, fixed, and stained with hematoxylin and eosin for histological examination.
We followed this procedure in exposing embryos to the following chemicals: benzene, toluene, and a mixture of the two. The concentrations of benzene and toluene in the ERS ranged from 10 parts per million (ppm) to 0.0001 ppm, and the concentrations in mixtures of benzene and toluene were in a similar range. Equal volumes of equal concentrations of benzene and toluene were combined to provide a series of 50-50 mixtures. As the concentration of benzene, toluene, and the mixtures increased, so did the mortality (Fig. 1A). In this preliminary study, the slopes of the curves in Figure 1A are similar. However, at the highest concentration (10 ppm), the slope of the combination of benzene and toluene declines; the significance of this effect will be determined in future experiments.
Dechorionated gastrula-stage embryos were immersed in different toluene concentrations for 30 min, and the lethal concentration that killed 50% of the animals (L[C.sub.50]) was found (0.1 ppm). Benzene, tested in this manner, had an L[C.sub.50] of 0.05 ppm, but the mixture of benzene and toluene had an L[C.sub.50] Of about 0.005 ppm. Therefore, the combination of benzene and toluene is acting synergistically; i.e., at intermediate doses, the mixture is more toxic to the embryos than either benzene or toluene alone (Fig. 1A). [TCDD (2,3,7,8,tetrachlorodibenzo-p-dioxin) and a mixture of TCDD and benzene have also been tested, and they display a similar synergism (pers. comm.)].
The retarded growth of treated embryos was immediately evident. Histopathological examination revealed many pycnotic nuclei in the organs of the treated fish when compared to the controls. Although multiple toxin-damaged nests of necrotic cells (often seen in acute toxicities) were lacking, multifocal death of individual cells was common and might represent the occurrence of apoptosis. The gills of these animals were poorly developed or even malformed; liver and gut also exhibited minimal differentiation.
Dechorionated zebrafish embryos exposed to benzene, toluene, or the mixtures also displayed specific cardiovascular defects. Histological observation of the heart, atrium and ventricle revealed a smaller tube with smaller and fewer cells than in the control animals. These embryos failed to reach the swim-up stage; their heart rate was greatly reduced, and they exhibited pericardial edema (Fig. 1B). Many of these animals lacked any visible circulation. In previous research, the microinjection of hexachlorobenzene into the perivitelline space of Japanese medaka gastrulae resulted in similar heart defects[3, 4]. Eighty-nine percent (89%) of the control zebrafish embryos -- untreated except for dechorionation -- developed at a normal rate and lacked cardiovascular defects (Fig. 1C).
These preliminary results indicate a need for further study of the effects of various combinations of mixtures of chemicals on embryonic development. The synergistic interaction of benzene and toluene adversely affecting embryonic development is in contrast to the "protective effect" of toluene in adult mice and humans when exposed to mixtures of benzene and toluene. This may indicate that during development the toluene-benzene mixtures affect embryos differently than if these fish were exposed to these chemicals as adults. It also raises the question of whether current mammalian transplacental toxicity monitoring is adequate. Additionally, the cardiovascular defects caused by benzene, toluene, and their mixtures are similar to those caused by hexachlorobenzene, dinitrophenol, parathion, carbaryl, tolbutamide, and 2,4,5-trichlorophenoxyacetic acid[3,4,6,7,8,9,10,11]. These results indicate that structurally diverse pollutants can have similar adverse effects on cardiovascular development.
The use of dechorionated zebrafish embryos in static exposure assays has provided a sensitive means for uncovering the effects of chemicals and chemical mixtures on early development. The mechanism of action of benzene or toluene and the basis for synergism have yet to be elucidated. The ability to perform genetic analysis combined with the rapidly increasing number of developmental genes that are being uncovered and characterized in the zebrafish make this system suitable for examination of molecular correlates of these defects. This zebrafish preparation should also prove useful in monitoring the environment for chemical pollutants.
Supported by grants to M.M. from the Department of Defense, #CBR-93DNA-2; Department of Energy, #DOE-EM-IFG0193E; and the United States Environmental Protection Agency, #CR822766-01-0; and by grants to J.J.S. from the Air Force Office of Scientific Research #(AFOSR)F49620-94-1-0368, and Sea Grant #NA46RG0470, R/P-60.
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