Chapter 11
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Evaluating in Vivo Toxicity of Chiral Pesticides Using the Zebrafish (Danio rerio) Embryo Model Chao Xu1 and Weiping Liu*,1,2 1Research Center of Environmental Science, College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, China 2Ministry of Education Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310029, China *E-mail:
[email protected] Enantioselectivity in toxicology of chiral pesticides has become one of the frontier topics facing toxicology. However, the toxicological data of chiral pesticides is in great scarcity. This is because of the different toxicological profiles between enantiomers and the racemates, and the dearth of enough enantiopure samples for toxicity assay. Zebrafish (Danio rerio) embryos provide an attractive model for determining the acute toxicity in relation to environmental risk assessment of chiral pesticides, offering the possibilities to perform small-scale, high-throughput analyses and less sample needs compared with other in vivo models. Beyond their application for determining acute toxicity, zebrafish embryos are also excellent models for understanding toxic mechanisms and the indication of possible adverse and long-term effects. Various applications of zebrafish embryos have been used for studying toxicogenomics, such as the effect of chemicals on gene and protein expression patterns. Other major possible applications include studies in metabolism, bioconcentration and biomarkers. These unique properties make them especially suitable for in vivo enantioselective toxicity assay of chiral pesticides.
© 2011 American Chemical Society In Chiral Pesticides: Stereoselectivity and Its Consequences; Garrison, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.
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Introduction Enantiomers of chiral pesticides have unique biological properties that make prediction of their toxicity very difficult. Their potential effects on human health and ecosystems are complex (1). Therefore, it is important for toxicological evaluation of chiral pesticides to include single enantiomer toxicological and safety evaluations in living bodies, so that actual risks of chiral pesticides are defined and adverse environmental consequences are minimized (2). On the other hand, recent advances in separation and analytical techniques have made possible the detection, isolation and preparation of enantiomers, although still on a small scale (3). Thus, there is an obvious need for the development of rapid, relevant and efficient testing strategies to evaluate the biological activity and toxic potential of chiral pesticides. In vitro studies such as those using cultured cells are a very important and productive approach to obtain understanding of toxic mechanisms of chiral pesticides, and in fact, they have been widely used. However, in vitro systems have the limitation of obtaining appropriate cell lines or primary cells and do not reflect the natural environment of cells in the body. Whole organism test studies can provide the most comprehensive understanding of toxic effects. However, the use of mammals is not only expensive, but also labor, time and dose-consuming. Furthermore, as the ethical concerns increasingly arise, the use of mammals in large scale toxicological screening programs has been limited. During recent decades, embryos and non-feeding larvae of the vertebrate zebrafish have been developed as cheap, effective alternatives that reduce and refine animal use in in vivo research (4–6). The zebrafish is a small tropical fish native to the rivers of India and South Asia (7). A number of unique features have contributed to its attraction in toxicology, such as its rapid development, easy maintenance in the laboratory, large number of offspring, transparency of embryos and access to experimental manipulation. After 24 hpf (hours postfertilisation), the basic body structure is laid out, and after approximately 2–3 dpf (days post-fertilisation), the embryos can hatch (Figure I). The transparent chorion enables easy observation of development stages (8). Zebrafish have a very short reproduction cycle. They reach maturity at the age of about 3 months. One female can spawn more than 100 eggs in each clutch which are fertilised by sperm release from the male into the water. Under good laboratory conditions several thousands embryo can easily be collected daily and used for parallel experimental treatments. These unique features make zebrafish a complete, developing vertebrate organism, and allow testing at levels of organ and organism toxicity. Also, their anatomic and genomic similarities with humans allow some predictability of mammal and human toxicity (9). On the other hand, early developmental life stages are often very sensitive to environmental insult, due to the enormous changes in cellular differentiation, proliferation and migration necessary to form required cell types, tissues and organs (6, 10). Since molecular signaling underlies all of these processes and most toxic responses result from disruption of proper molecular signaling, early developmental life stages are the ideal stage to determine toxicology of chiral pesticides. 168 In Chiral Pesticides: Stereoselectivity and Its Consequences; Garrison, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.
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Figure I. Zebrafish embryos at different development stages. The zebrafish embryo was originally used to study the genetics of development due to its transparency, quick embryonic development, easy collection in high numbers and similarity with human development (11). Nowadays, zebrafish offer a model with basic structure not much different from a mouse or a human (12). It allows not only phenotypic screens to identify gene function on a large scale, but offers many mutation types for research (12, 13). These mutagenesis protocols are complemented by reverse genetic techniques that allow manipulation of specific gene functions (14). In recent years, small chemicals were induced to manipulate the specific developmental pathways of embryos (e.g. inhibitors of Fibroblast Growth Factor, Retinoic acid and Sonic Hedgehog) in a conditional manner (15–17). Use of zebrafish embryos has the unique advantages of abundance background knowledge, technology and approaches.
Zebrafish Maintenance and Egg Production In our studies, zebrafish were maintained in a light/dark cycle of 14:10 h at 26±1 ºC under semi-static conditions with charcoal filtered water. The zebrafish were fed with live brine shrimp (Artemia nauplii) twice a day. Since our primary aim was to reproducibly produce large amounts of eggs for exposure studies, we also used commercially available pet shop fish and customized the standard procedures according to our needs. Briefly, embryos were obtained from adult fish free of macroscopically discernable symptoms of infection and disease with a preferable ratio of 1:2 for female to male. Four groups of genitors were placed 169 In Chiral Pesticides: Stereoselectivity and Its Consequences; Garrison, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.
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separately in a specific spawning aquarium equipped with a mesh bottom to prevent the eggs from being cannibalized. Spawning was induced in the morning when the light was turned on. Half an hour later, eggs from each aquarium were collected and used immediately for the exposure experiment. For details about the maintenance, feeding and breeding of zebrafish see the Zebrafish Information Network ZFIN (Zebrafish Information Network). Zebrafish embryos are so small that several of them can even be placed in the well of a 384 well plate. This makes it possible to achieve medium to high throughput toxicity testing. In some recent studies, such systems show the ability to match the chemical entitles with specific biological activity. During recent years, automated screening technologies with zebrafish embryos (i.e. microscopes combined with intelligent image acquisition systems) have been extensively developed, these provide toxicological profiles of toxicants at high spatial and temporal resolution.
Applications of the Zebrafish Model Acute Toxicity and Teratogenicity Acute fish toxicity tests are widely required for the testing of pesticides for ecological risk assessment (18, 19). However, as animal welfare legislations becomes more and more important, there is a great public demand for the replacement of animal tests for ethical reasons. Also, less time consuming and expensive replacement of testing methods are required. The zebrafish embryo (up to 3-4 days after hatching) is not considered an animal by current European legislation (20), and therefore, its use in research studies is coherent with trying to reduce and replace the use of rodents for toxicity studies. As a matter of fact, the first application of the zebrafish embryo in environmental research was promoted by the aim to develop an alternative to the 96-h acute fish toxicity test (21). Acute toxicity in fish embryos correlates very well with acute toxicity in adult fish.And fish embryo test has been submitted to the Organization for Economic Co-operation and Development (OECD) (22). In Germany, the zebrafish embryo test was introduced as a standardized ISO assay replacing traditional toxicological tests with adult fish (21, 23). In our preliminary study with the synthetic pyrethroids of lambda-cyhalothrin, the enantiomers showed the same enantioselective toxicity as in the fish toxicity test, that is, the (−)-enantiomer was more than 100 times toxic than the (+)-enantiomer in 96-h acute toxicity test with zebrafish adults (Table I). The zebrafish embryo acute toxicity test showed the same pattern; the (−)-enantiomer is 7.2 times stronger in 96-h mortality (24). In a study with fenvalerate, the αS-2S-enantiomer was 56 times more toxic than the αR-2R-enantiomer (25). The assay of zebrafish embryo-larval showed that the αS-2S-enantiomer was 3.8 times stronger than the αR-2R-enantiomer in 96-h mortality. Screening for developmental disorders as an indicator of teratogenic effects (such as crooked body) can also be included in analysis of acute toxicity in embryos (21, 26). Other morphological, sublethal endpoints, such as heart beat rates, yolk sac edema and spontaneous movements may also supply useful 170 In Chiral Pesticides: Stereoselectivity and Its Consequences; Garrison, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.
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information of long-term effects of chemicals. In our study with acetofenate, significant enantioselectivity in developmental toxicities such as yolk sac edema and pericardial edema was observed in zebrafish embryo (27). These pattern followed the order (+)-enantiomer > (±) –racemate > (−)-enantiomer. Endpoints include yolk edema, pericardial edema and crooked body. In the study with lambda-cyhalothrin, significant difference was observed between the two enantiomers (27) (Table II). The mortality of zebrafish embryo test showed that the (−)-enantiomer was 7.2 times stronger than the (+)-enantiomer in mortality. The teratogenicity also showed the similar patterns. Statistical analysis showed that the (−)-enantiomer was significantly more toxic than the (+)-enantiomer in inducing crooked body and pericardial edema. In other study with fenvalerate, four stereoisomers showed enanioselectively embryonic toxicity in inducing crooked body, yolk sac edema and pericardial edema (25). The αS-2S-isomer was most toxicity in developmental toxicity of zebrafish embryo. Although most assays using zebrafish embryos rely on morphological endpoints, chiral factors are implicated in the actual teratogenic mechanism of several compounds. In order to further reveal the toxicant-specific mechanisms of action, the gene expression profile should be involved in studies.
Table I. LC50 values of enantiomers of lambda-cyhalothrin for aquatic vertebrate zebrafish (µg·-1). Ref. (24) Exposure time
(±)
(-)
(+)
24h
2.123±0.062
2.033±0.042
>120
48h
1.105±0.034
1.025±0.034
>120
72h
0.832±0.021
0.794±0.021
>120
96h
0.875±0.032
0.740±0.032
>120
Endocrine Disruption A wide variety of pesticides have been shown to mimic endogenous hormones. The presence of these compounds in the aquatic environment has been associated with a number of reproductive disorders or disruption of sexual differentiation, particularly in fish (28). These chemicals induce endocrine disruption act via estrogen, androgen and thyroid receptors. By measuring the effect of exposure on hormone-responsive genes, the zebrafish embryo model can be an important screening tool for endocrine disrupting compounds. Such a screening assay has been proven to have as much sensitivity as other endpoints and screening systems (29, 30). Furthermore, it is principally possible to monitor of other endpoints like androgenic, glucocorticoid, thyroid or other hormone markers in the embryo model.
171 In Chiral Pesticides: Stereoselectivity and Its Consequences; Garrison, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.
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Table II. Summary of enantioselective embryo toxicity of lambda-cyhalothrin responsive endpoints (mg L-1)) 48-h
Developmental defects
NOECsd
96-h
±
+
-
±
+
-
±
+
-
a
a
a
1.30
a
1.30
1.30
a
0.18
crooked body
0.07
c
0.16
0.03
0.46
0.03
0.03
0.09
0.03
yolk sac edema
0.33
b
b
b
b
>0.30
b
b
b
pericardial edema
0.30
d
>0.30
0.07
>0.30
>0.30
0.05
0.34
0.09
crooked body
