ToxSci Advance Access originally published online on March 28, 2008
Toxicological Sciences 2008 104(1):177-188; doi:10.1093/toxsci/kfn065
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Development of a New Screening Assay to Identify Proteratogenic Substances using Zebrafish Danio rerio Embryo Combined with an Exogenous Mammalian Metabolic Activation System (mDarT)
* Institute of Toxicology, Merck KGaA, 64293 Darmstadt, Germany
Institute of Hydrobiology, TU Dresden, 01062 Dresden, Germany
1 To whom correspondence should be addressed at Merck KGaA, Institute of Toxicology, Building U9, Frankfurter Str. 250, 64293 Darmstadt, Germany. Fax: +49-(0)-6151-72-912173. E-mail: thomas.broschard{at}merck.de.
Received February 6, 2008; accepted March 14, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGLOSSARYREFERENCES |
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The assessment of teratogenic effects of chemicals is generally performed using in vivo teratogenicity assays, for example, in rats or rabbits. We have developed an in vitro teratogenicity assay using the zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (MAS), able to biotransform proteratogenic compounds. Cyclophosphamide (CPA) and ethanol were used as proteratogens to test the efficiency of this assay. Briefly, the zebrafish embryos were cocultured at 2 hpf (hours postfertilization) with the test material at varying concentrations, induced male rat liver microsomes and nicotinamide adenine dinucleotide phosphate (reduced) for 60 min at 32°C under moderate agitation in Tris-buffer. The negative control (test material alone) and the MAS control (MAS alone) were incubated in parallel. For each test group, 20 eggs were used for statistical robustness. Afterward fish embryos were transferred individually into 24-well plates filled with fish medium for 48 h at 26°C with a 12-h light cycle. Teratogenicity was scored after 24 and 48 hpf using morphological endpoints. No teratogenic effects were observed in fish embryos exposed to the proteratogens alone, that is, without metabolic activation. In contrast, CPA and ethanol induced abnormalities in fish embryos when coincubated with microsomes. The severity of malformations increased with increasing concentrations of the proteratogens. We conclude that the application of microsomes will improve and refine the D. rerio teratogenicity assay as a predictive and valuable alternative method to screen teratogenic substances.
Key Words: zebrafish; cyclophosphamide; ethanol; alternatives to animal testing; teratogenicity; metabolic activation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGLOSSARYREFERENCES |
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The embryotoxic and teratogenic potential of chemicals is mainly investigated in animals, for example, in rats and rabbits. There are few alternative methods investigating the embryotoxicity and/or teratogenicity of substances. In the seventies, the rat whole-embryo culture was first proposed by New (1978
; Webster et al., 1997) followed later on with other animal models (e.g., mouse, rabbit). In the late eighties, cell and organ culture systems completed this toolbox. However, it is highly disadvantageous that some of those models (1) still need animals to obtain the test systems (Piersma, 2004); (2) in general do not cover the whole period of embryo-/fetogenesis; and (3) do not address a possible metabolic activation of the test materials (Spielmann et al., 2006).
It is well established that teratogenic activity is not always due to parent compounds but may be caused by metabolites formed by maternal metabolism (Fantel, 1982; Webster et al., 1997). Parent compounds, termed proteratogens, can be bioactivated to highly teratogenic metabolites, for example, electrophiles or free radical intermediates (Wells et al., 2005). Therefore, the addition of a mammalian metabolic activation system (MAS), such as S9-mix, microsomes, hepatocytes, to detect proteratogenic potency has been proposed using whole-embryo systems (Fantel et al., 1979, Zhao et al., 1993).
The zebrafish Danio rerio embryo, a vertebrate model, combines the advantages of cell culture (easy handling, no animal testing) and embryo culture systems (complex test system). This model is ideally suited to study the fundamental processes underlying embryonic development. The development of the zebrafish is very similar to the embryogenesis in higher vertebrates, including humans. A single female can lay up to 400 eggs per week (Laale, 1977) and spawn throughout the year under laboratory conditions. Zebrafish embryos develop outside the female body and moreover, the shell of their eggs (chorion) is completely transparent enabling the detailed observation of the developing embryo very easily (Fig. 1). Zebrafish embryonic development is very rapid: At 24 hpf (hours postfertilization) all major organs are formed and at 72 hpf the fish hatch (Dahm and Geisler, 2006). The different stages in the development of the zebrafish until hatching are studied in detail and summarized elsewhere (Kimmel et al., 1995). These advantages make the zebrafish an ideal model organism to study various aspects of the developmental processes in vertebrates.
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In the last 40 years, extensive studies have been performed on this in vitro model. Amongst others, zebrafish D. rerio embryo has been promoted as a general model for assessing embryotoxicity and teratogenicity using the so-called DarT assay (Zebrafish D. rerio Teratogenic assay). A scoring system was described for DarT to assess compounds that trigger teratogenicity (Bachmann, 2002
; Nagel, 2002). However, the absence of metabolic systems in the in vitro assays is a major limitation to their applicability (Spielmann et al., 2006). In this paper we describe the successful combination of DarT with a MAS (microsomes), which is presented as a new alternative method for complete developmental toxicity testing, named mDarT. For this purpose, two referenced proteratogens, cyclophosphamide (CPA), and ethanol were investigated.
CPA is an alkylating antineoplastic agent in various therapeutic categories. The drug prevents cell division by cross-linking DNA strands and decreasing DNA synthesis. The mechanism by which this occurs is apparently through its metabolites phosphoramide mustard and acrolein (Schardein and Macina, 2007). It is well known, that liver microsomes are able to transform CPA to alkylating and cytotoxic metabolites (Torkelson et al., 1974). In rats, CPA is oxidatively activated by the CYPs isozymes CYP2C6/2C11 and CYP2B1 (Gut et al., 2000). Phenobarbital (PB) and β-naphthoflavone (βNF) are well-known inducers for these CYPs in the rat. Therefore, PB and βNF-induced rat liver microsomes were used for this study to bioactivate CPA.
Ethanol acts as a central nervous system depressant with intoxicating properties (Schardein and Macina, 2007). Its developmental toxicity was first documented in human fetuses in 1968. Fetal alcohol syndrome was rediscovered in 1973 by Jones and Smith and refers to a pattern of defects in the offspring of alcoholic women (Abel and Dintcheff, 1978; Jones and Smith, 1973). Although the teratogenic properties of ethanol have been firmly established, the underlying mechanism(s) of toxicity remains unclear. Two molecular mechanisms have been postulated, which include direct ethanol effects and the indirect effects associated with ethanol metabolism, such as acetaldehyde formation and oxidation stress (Reimers et al., 2004). Rat microsomal fractions are able to metabolize ethanol. Enzymes involved are CYP2E1, P450 reductase and others (Quintans et al., 2005). Isoniazid (INH) is a well-known inducing agent for CYP2E1. Therefore, INH-induced rat liver microsomes were used for this study.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGLOSSARYREFERENCES |
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Animal Care and Egg Production
A breeding stock of unexposed and healthy mature zebrafish (supplier: Aquarium West, Germany; breeder: Institute of Toxicology, Merck) older than 6 months was used for the egg production. Spawners were maintained in aquaria at 26°C with a loading capacity of a minimum of 1 l of water per fish. Lighting was controlled by a timer to provide a 12-h light/dark cycle. Females and males were continuously held together in a ratio of 1:2. Dry flake food was fed twice a day and live food (white mosquito larvae) was fed once a day (Organization for Economic Cooperation and Development, 2006
; Westerfield, 2000).
Mating and spawning took place within 15 min after turning on the lights in the morning. To prevent adult zebrafish from egg predation, eggs traps were covered with a stainless steel mesh with a grid size of 2 mm. Plastic plant imitations serving as spawning substrate, were fastened onto the mesh. About 20–30 min after the onset of light the eggs traps were removed and the eggs were collected (Organization for Economic Cooperation and Development, 2006). At the culture conditions described above, fertilized eggs undergo the first cleavage after approximately 15 min and consecutive synchronous cleavages form 4, 8, 16, and 32 cell blastomeres. At this stages (4–32 cells) eggs can be identified clearly as fertilized and only these can be used for the experiment (Nagel, 2002; Schulte and Nagel, 1994).
Materials
Tris-HCl and ethanol were purchased from Merck KGaA (Germany). β-nicotinamide adenine dinucleotide phosphate reduced form (NADPH) and 3-aminobenzoic acid ethyl ester methanesulfonate (MS-222) were obtained from Sigma (Germany). CPA monohydrate was delivered from Calbiochem (Germany). The induced rat liver microsomes (INH or the mixture βNF and PB) were provided by Tebu-bio (Germany). The fish medium, that is, reconstituted water consisting of 2mM CaCl2, 0.5mM MgSO4, 0.7mM NaHCO3, and 0.07mM KCl, was prepared in the test facility (Organization for Economic Cooperation and Development, 2006). All solutions were freshly prepared and chemicals were dissolved one hour before incubation. NADPH and CPA were dissolved in Tris-buffer. From the substances cited, hazardous properties may not be excluded. Therefore, chemicals or biological materials must be handled with the appropriate care.
Methods
Temperature.
The optimal temperature of mammalian microsomes is 37°C. In contrast, fish embryos develop optimally at a temperature of 26°C–28.5°C. However, they tolerate an eight degree range, between 25°C and 33°C. Incubating them for long periods above or below these extremes may produce abnormalities (Kimmel et al., 1995). Thus, mammal microsomes were incubated at 32°C to achieve the highest metabolic activation together with normal fish embryo development.
Metabolic activation system.
Tris was dissolved in fish medium and neutralized with HCl. The Tris-HCl buffer concentration was 0.1M, pH 7.6 at 25°C. Microsomes were diluted in Tris-buffer. The induced rat liver microsomal protein final concentration was 0.7 mg/ml assayed by the bicinchoninic acid protein assay (Smith et al., 1985). The NADPH final concentration was 1mM as recommended by the supplier. The final volume of the reaction was 2 ml.
Embryo exposure.
For each test item a concentration range finding experiment was conducted (three to four concentrations) with a constant spacing factor of 2. In general, two controls were used in each experiment: (1) a negative control consisting of the highest concentration of the proteratogen without MAS and (2) the MAS control consisting of the MAS alone. Typically, 20 fish embryos were used per concentration group.
Fish embryos were washed twice in glass Petri dishes with Tris-buffer (see Fig. 2). Within the first hour after fertilization of the eggs (1 hpf), the fecundated fish embryos (from 4 to 32 cell blastomeres) were sorted under a stereomicroscope (Zeiss, Germany) and collected in a plastic Petri dish containing Tris-buffer. From the pooled fecundated fish embryos, 20 eggs were randomly transferred into a 2-ml vial (Eppendorf) for each group. All these steps were performed at 26°C.
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Subsequently, the MAS was prepared in a 16-mm polypropylene test tube (BD-biosciences, Germany) stored on ice, filled with Tris-buffer, microsomes and the test material (i.e., premix). A 5-min preincubation step was performed at 32°C and 100 rpm for the test tubes containing the premix and the NADPH stock solution (10mM). Afterward, 20 fish embryos were transferred together in 800 µl into a test tube using a pipette and, thereafter, NADPH was added to start the reaction at the latest 2 hpf. The incubation was performed for 60 min in a shaking water bath (GFL-mbH, Germany) at 32°C, 100 rpm.
Thereafter, the exposure was stopped by transferring the fish embryos into different Petri dishes filled with fish medium (one Petri dish per group). Afterward, the fish embryos were cultured individually in 24-well plates (Nunc GmbH, Germany) containing 2 ml of fish medium per well and left for 48 h at 26°C with a 12-h light/dark cycle in a precision incubator (Memmert GmbH & Co. KG, Germany). Developmental parameters were monitored and documented (see below). After 48 hpf, the fish embryos were anesthetized and killed using a MS-222 solution (0.3%).
Scoring.
At different time points, that is, 8, 24, and 48 hpf, the fish eggs were evaluated and scored for lethal or teratogenic effects using an inverted microscope with phase contrast optics, a mounted time-lapse recorder and the analysis software (Olympus PA). All embryos were staged as previously described (Nagel, 2002). The different lethal or teratogenic endpoints are summarized in Table 1. The effects observed were documented under the Quality Management System established at the Merck preclinical development division.
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Validity parameters and statistics.
The fertilizing rate of the fish eggs should be higher than 50% (DIN 38415-6, 2003
). The assay is considered to be valid if the viability of the control eggs (MAS and negative) exceeds or is equal to 90% after 48 hpf (no teratogenic or lethal effects). A one-way ANOVA test was run and a Tukey's post hoc was performed when significant differences were reported.
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RESULTS |
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Cyclophosphamide
Controls.
Control embryonic stages were shown in Figure 1. As summarized in Tables 2 and 3, the negative control (CPA without MAS) triggered no lethal effects in the fish embryos and only one of 60 fish eggs displayed malformations (head) within three experiments (2% of surviving eggs). In the three MAS control experiments (MAS alone), three fish embryos were coagulated (5% of all eggs) and one fish embryo displayed malformations of the sacculi/otoliths and the end tail (2% of surviving eggs) (Table 3). The total percentage of teratogenic and/or lethal eggs in each individual control group experiment consisting of 20 eggs each (negative or MAS control) was
10% after 48 hpf and, thus, all experiments were considered to be valid.
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Lethal and/or teratogenic effects of metabolic activated CPA in D. rerio embryos.
The lethal and/or teratogenic effects observed at 48 hpf, which were induced in fish embryos incubated with both CPA and rat liver microsomes, are summarized in Tables 2–4
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Table 2 summarizes the lethal effects observed in fish eggs incubated with different concentrations of metabolized CPA. There were no or only five coagulated eggs of 60 fish eggs at 3.5, 7.0, and 14.0mM CPA, respectively. However, in all three single experiments a significant increase of coagulated fish eggs was observed 48 hpf at the highest concentration 28mM (41 of 60 fish eggs). In this group a strong increase in the incidence of coagulated eggs was observed between 24 and 48 hpf.
In the test groups containing both MAS and CPA, the percentage of teratogenic eggs (teratogenic eggs/surviving eggs) clearly increased with raising CPA concentrations reaching significance at 14 and 28mM (p < 0.01) as summarized in Table 3. The absolute number of fish eggs displaying teratogenic effects increased from 4 (3.5mM CPA) to 34 (14mM CPA). At the highest concentration (28mM CPA) the lethal effects were predominant, that is, 41 from 60 fish eggs were coagulated. Thus, the absolute number of teratogenic fish eggs in the highest concentration group drops down, however, all 19 surviving fish embryos of this group displayed teratogenic effects (100% teratogenic eggs).
The absolute and relative incidences of the different CPA-induced teratogenic endpoints in zebrafish are summarized in Table 4 for the different control and test groups. With the exception of rachischisis all possible malformations listed in Table 1 have been observed in fish embryos treated with metabolized CPA. In few cases some slight findings observed at 24 hpf were not observed at 48 hpf. These findings were shown to be reversible, which is, however, not uncommon for biological systems.
Some malformations such as effects on the head occurred more frequently than others. Teratogenic effects were considered as "fingerprint endpoints" if the following criteria were fulfilled: (1) concentration-response relationship and (2) the endpoint must be observed in 
50% of all teratogenic fish eggs in the test groups. As 67 fish eggs exposed to CPA displayed one or more teratogenic effects, a fingerprint-endpoint must be observed in at least 34 fish eggs within the three experiments (Table 4).
Following these criteria, the major endpoints are shown in Figure 3. Malformations of the head (Figs. 3A, 3B, and 3D), the sacculi/otoliths (Figs. 3A–D), and the spinal cord structure (Figs. 3C and 3D), are defined as fingerprint malformations of CPA in fish embryos. Other malformations such as effects on the end tail (Fig. 3C) or scoliosis (Figs. 3B and 3C) were also observed frequently, however, they did not fulfill the criteria for fingerprint endpoints.
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Figure 4 illustrates CPA-induced teratogenic effects depending on the concentration of CPA and on the anatomic region within the fish embryo. Figures 4A–C display frontal, axial and ventral malformations, respectively. Figure 4D presents growth retardation, that is, the developmental stages of the test groups are compared with the control groups.
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A clear concentration-response relationship was obtained for all different malformations observed at the frontal (Fig. 4A), axial (Fig. 4B), and ventral (Fig. 4C) zones (see also Table 4), that is, the incidence of malformations increased with increasing concentrations of CPA. In contrast, a clear incidence of growth retardation (Fig. 4D) was only observed in the test group with the highest CPA concentration.
Taken together, a clear concentration-response relationship was observed for both teratogenic and lethal effects in D. rerio fish eggs exposed to CPA and a mammalian metabolic system. Significant increases (p < 0.01) in teratogenic effects compared with the controls were observed in test groups exposed to 14 and 28mM CPA. Moreover, a significant increase (p < 0.01) in lethality was observed in the highest concentration group (28mM CPA). Three fingerprint malformations (effects on the head, sacculi/otoliths and spinal cord structure) were identified in the fish embryos.
Ethanol
Controls.
As shown in Tables 5 and 6, only one coagulated fish egg (2% of all fish eggs) and four fish eggs displaying teratogenic effects (7% of surviving embryos) were obtained in three negative control experiments each consisting of 20 fish embryos exposed to 170mM ethanol without MAS. In the three MAS control experiments (MAS alone), no teratogenic effects were observed, but four of 60 fish eggs were coagulated (7% of all fish eggs). However, for each control group and for the three experiments, the acceptance criterion of 10% of impaired embryos after 48 hpf was satisfied and, thus, the experiments were considered to be valid.
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Lethal and/or teratogenic effects of metabolic activated ethanol in D. rerio embryos.
Lethal and/or teratogenic effects observed at 48 hpf in fish embryos incubated with both ethanol and rat liver microsomes, are summarized in Tables 5–7
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Table 5 summarizes the lethal effects observed in fish eggs incubated with different concentrations of metabolized ethanol. Only five and three coagulated eggs out of 60 eggs were observed in the test groups exposed to 42.5 and 85mM ethanol, respectively. However, a significant increase of coagulated fish embryos (22 of 60 fish eggs) was observed after 48 hpf at the highest concentration of 170mM ethanol.
In the test groups, the incidence of eggs displaying malformations (teratogenic eggs/surviving eggs) increased with increasing ethanol concentrations reaching significance at 85 and 170mM (p < 0.05) as summarized in Table 6. The absolute number of fish eggs displaying teratogenic effects was 6, 14, and 17 at 42.5, 85, and 170mM ethanol, respectively, that is, the absolute number of teratogenic eggs was comparable for the two highest ethanol concentrations. However, the incidence of teratogenicity (teratogenic eggs/surviving eggs) increased with increasing ethanol concentrations, as the lethality was much higher at 170mM ethanol (22 coagulated eggs) compared with 85mM ethanol (three coagulated eggs).
The absolute and relative incidences of the different ethanol-induced teratogenic endpoints in zebrafish are summarized in Table 7. With the exception of rachischisis all possible malformations listed in Table 1 have been observed in fish embryos treated with metabolized ethanol. As with CPA, some slight findings observed at 24 hpf were shown to be reversible after 48 hpf.
According to the above-mentioned definition of fingerprint malformations, the major endpoints are shown in Figure 5. Effects on the sacculi/otoliths (Figs. 5A–C), the spinal cord structure (Figs. 5A–C), and the head (Figs. 5A and 5C) as well as scoliosis (Figs. 5B and 5C) were the predominant ethanol-induced malformations in D. rerio embryos ( 50% of all teratogenic fish eggs in the test groups).
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In Figure 6, the teratogenic effects are presented depending on the ethanol concentrations and the anatomic region within the fish embryo. Figures 6A–C display frontal, axial, and ventral malformations, respectively. Figure 6D summarizes growth retardation.
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A clear concentration-response relationship was obtained for all teratogenic effects at the frontal (Fig. 6A) and ventral (Fig. 6C) zones. In addition, a concentration-dependent retardation of fish embryo development was observed (Fig. 6D). The incidence of most malformations of the axial region increased with increasing concentrations of ethanol. However, incidences of end tail defects (Fig. 6B) were not concentration dependent and again no rachischisis was observed (Fig. 6B).
Based on these three studies, a clear increase of teratogenic incidences was observed with increasing concentrations of metabolic activated ethanol reaching significance (p < 0.05) at 85 and 170mM ethanol, respectively. In addition, lethality was significantly increased (p < 0.05) at the highest test material concentration (170mM ethanol). Moreover, four characteristic malformations (effects on the sacculi/otoliths, cordal structure and head as well as scoliosis) were identified in fish embryos exposed to metabolically activated ethanol.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGLOSSARYREFERENCES |
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In this study we demonstrated the successful combination of the fish embryo assay, used for screening of embryotoxic/teratogenic effects, with a mammalian MAS. In addition, we showed hereby that there is insufficient CYP activity in the zebrafish embryos at the earlier stages (2–3 hpf) to bioactivate proteratogens, for example, CPA. Indeed, Cytochrome P450s (CYPs) have been detected in zebrafish embryos at 24, 72, and 120 hpf (Wang et al., 2004
; Wang-Buhler et al., 2005; Tseng et al., 2005). However, currently there is neither qualitative nor quantitative proof that CYPs are expressed at earlier stages of D. rerio development. Rat liver microsomes were chosen as the MAS as they exert less toxicity to fish embryos compared with other MAS, for example, primary hepatocytes (data not shown).
The efficiency of this advanced in vitro screening model was demonstrated using the two human proteratogens CPA and ethanol as model compounds. As mentioned above, it was shown that exposure of fish embryos to each proteratogen alone did not result in any significant teratogenic effect. In contrast, only the presence of the mammalian MAS triggered the formation of significant lethal or teratogenic effects in exposed fish embryos.
With the exception of rachischisis all teratogenic endpoints listed in Table 1 were observed in fish embryos exposed to metabolically activated CPA. The incidences of lethality and most teratogenic effects were clearly concentration-dependent. The major malformations, that is, the teratogenic endpoints with the highest incidences, comprised effects on the head, the sacculi/otoliths and the spinal cord structure. As already mentioned, these effects were defined as fingerprint endpoints in fish embryos according to the definition provided above. Like many other antineoplastic agents, CPA elicited a full spectrum of developmental toxicity in mammals like mice (Hackenberger and von Kreybig, 1965), rats (Murphy, 1962) and rabbits (Gerlinger and Clavert, 1964) including growth retardation (reduced fetal body weight) and effects on the limb, digit, palate and jaw when administered intraperitoneally during organogenesis at dose levels of 2–50 mg/kg/day. In humans, a specific syndrome of defects was identified with CPA including congenital malformations of the digit, palate, ears, face, skin and in some cases the skull (Schardein and Macina, 2007). Thus, some of the major effects observed in fish embryos exposed to metabolically activated CPA are similar to characteristic anomalies observed in mammals and humans.
As with CPA, exposure to ethanol without an external MAS was insufficient to induce any significant developmental malformations in fish embryos up through a concentration of 170mM. Only the concomitant use of ethanol and microsomes provoked lethality and teratogenicity starting at the lowest concentration (42.5mM). As demonstrated by other groups, ethanol is able to induce malformations in fish embryos without a MAS and, thus, acting as a direct teratogen in this test system (Reimers et al., 2004, 2006). However, these teratogenic effects were observed only at higher ethanol concentrations ( 200mM) and/or increased incubation times. In fish eggs exposed to ethanol and microsomes, all malformations with the exception of rachischisis were observed with different incidences. Based on the definition of fingerprint endpoints, effects on the spinal cord structure, the sacculi/otoliths, the head as well as scoliosis were the major malformations observed. The major malformations induced in fish embryos by lower concentrations of bioactivated ethanol match with the effects observed in fish eggs exposed either to higher concentrations of ethanol or its major metabolite acetaldehyde (Reimers et al., 2004). It is well known, that ethanol is teratogenic in animals and humans: In vivo studies in several species (e.g., mice, rats, rabbits, guinea pigs) clearly demonstrated potent developmental toxicity including retarded fetal growth, increased mortality and malformations. In humans, the most typical abnormalities are associated with the central nervous system, the craniofacial development, the cardiac system and the skeleton (Schardein and Macina, 2007). Again, major ethanol-induced effects observed in the fish embryos are similar to effects observed in ethanol exposed mammals and especially in humans: The malformations of the head and the spinal cord observed in fish embryos correspond to the craniofacial and skeletal abnormalities in humans.
Based on the results presented it could be shown that only the combination of fish embryos with a MAS was able to detect the teratogenic potential of the proteratogenic compounds CPA and ethanol. With both compounds a clear concentration-response relationship was demonstrated and the qualitative nature of major malformations was similar between fish embryos and mammals.
Numerous advantages make this model an excellent alternative method to the in vivo teratogenicity assay, that is, (1) short time of the assay based on the rapid embryonic development (48 hpf, from the oviposition to the end of the test); (2) a huge amount of fish eggs daily available to perform screening studies; and (3) the ease to apply the technique in facilities.
However, despite these advantages some limitations of this system have to be taken into a account: (1) The chorion membrane enclosing the fish embryo might limit the absorption of very lipophilic substances or substances with a high molecular weight making it difficult to detect their proteratogenic potential; (2) due to the toxicity of the microsomes to the fish embryos the contact time with the test material is limited to 1 h, that is, the fish embryo is exposed to the test item only during a small part of the developmental period. Although the exposure time covers the most sensitive phase of the developmental period in the current protocol as demonstrated with CPA and ethanol, some proteratogens, which are active at a later developmental stage might be missed. (3) The CYP P450 activity of liver microsomes depends on the inducing compound, with which the rats were treated. In general, the use of microsomes exhibiting a broad range of CYP activities is preferred (e.g., by using PB/βNF-induced microsomes). However, proteratogens activated by a specific CYP P450 isoenzyme (e.g., CYP 2E1) might exhibit lower activity or might be even missed when using generally induced microsomes. Therefore, the application of different liver microsomes from rats treated with different inducers should be considered depending on the test material.
With the mDarT assay we present a powerful and sensitive in vitro model for the detection of teratogenic and proteratogenic substances as shown with two proteratogens, CPA and ethanol. Further proteratogens are currently being investigated with mDarT to evaluate the power of this alternative method. In addition, characterization of the metabolites formed in this test system might offer more insights in the teratogenic mechanism of proteratogens. In the future, a close collaboration between scientists from academia, industry and regulatory authorities may allow a prevalidation process and in the long-term even a validation and the partial replacement of animal testing for teratogenicity.
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GLOSSARY |
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- Coagulation. Denatured fish egg. No clear structures of the fish embryo are observable anymore.
- Heart structure in the zebrafish Danio rerio. At 48 hpf, in fish embryos, the normal looping process places the ventricle and atrium side by side, so that the two chambers largely overlap each other in lateral view (Antkiewicz et al., 2005
).
- Sacculi/otoliths. This is the primitive ear structure in the zebrafish D. rerio embryo. The zebrafish possess two otoliths per sacculi. The otoliths are dark round bones within the sacculi (or vesicle). The otolith malformations included none or one to multiple otoliths per sacculi. The sacculi malformations included abnormally shaped vesicles (Reimers et al., 2004
).
- Somites. Undifferentiated mesodermal component of an early trunk or tail segment or metamere, derived from paraxial mesoderm; forms the myotome, the sclerotome and perhaps the dermatome (Kimmel et al., 1995
).
- Yolk. Nutrient store for embryonic development in the form of semicrystalline phospholipoprotein and contained within yolk granules (Kimmel et al., 1995
).
- Yolk sac. Giant syncitial uncleaved cells containing the yolk; underlies the blastodisc early, and becomes enveloped by the blastoderm during epiboly (Kimmel et al., 1995
).
- Yolk sac extension. The posterior elongated region of the yolk sac that forms during the segmentation period (Kimmel et al., 1995
).
- Heart structure in the zebrafish Danio rerio. At 48 hpf, in fish embryos, the normal looping process places the ventricle and atrium side by side, so that the two chambers largely overlap each other in lateral view (Antkiewicz et al., 2005
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ACKNOWLEDGMENTS |
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We wish to thank Gerd Ziegler and Andreas Gado for excellent technical assistance.
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