Abstract
We are confronted with a large and steadily growing number of bioactive compounds, including drugs, pesticides and industrial by-products. The assessment of target specificity and potential toxic effect on human health and the environment generates a strong demand for robust and cost-effective models with high predictive power. Here, we investigate the potential of the zebrafish embryo as a whole-organism, vertebrate model to assess the specificity of compounds that are known to inhibit Acetylcholinesterase (AChE). Inhibitors of AChE are widely used as drugs and pesticides. By application of simple assays and comparison with the phenotype of embryos with genetic lesions in the ache gene, we demonstrate that only one of the AChE inhibitors (galanthamine) reproduces the phenotype of ache mutant embryos. The other compounds produced additional effects indicating secondary targets. Our work demonstrates the power of a genetic system for toxicological evaluations.
The combination of genetics and transgenesis with the other experimental virtues of the zebrafish embryo, such as small size and low cost, offers a whole organism platform for medium to high throughput compound testing.
Introduction
A powerful methodology in development of bioactive chemicals has arisen from the synergy between structural information on the target molecules and combinatorial chemistry (Norin and Sundstrom, 2001) . While the capability to synthesise novel compounds increased enormously in recent years, technologies to assess the biological activity and target specificities of these novel molecules did not match the pace of the modern synthesis capabilities (Balls, 2002) . A good drug is potent and highly specific with a well-defined target. Ideally, the phenotypic consequences of an inhibitory compound should be the same as that of a genetic mutation causing loss-of-function of the drug target. Simple genetic models, such as yeast (Marton et al., 1998) or C. elegans (van Kessel et al., 1989 ) may help to identify primary and secondary targets but they are too distant in evolutionary time to provide a comprehensive toxicological profile for vertebrates.
Besides attracting ethical concern, the use of mammals is expensive and labour intensive, limiting an application in large-scale screening programs. Embryos and larvae of the lower vertebrate zebrafish (Danio rerio) may offer a cheap and effective alternative.
Zebrafish embryos/larvae are small and can be obtained in large numbers throughout the year at a fraction of the cost of a mouse or a rat. Zebrafish embryos develop outside of the mother allowing systematic compound screens (Peterson et al., 2000) from earliest life stages onwards. Moreover, a large number of mutations affecting signalling pathways and physiological processes relevant to human pathology and pharmacology are available (Barut and Zon, 2000; Driever et al., 1996; Haffter et al,. 1996; Langheinrich et al., 2002; Stern and Zon, 2003) .
We have investigated here the application of wildtype and mutant zebrafish embryos in drug testing by phenotypic comparisons with a mutation in the acetylcholinesterase (ache) by guest on January 11, 2016 http://toxsci.oxfordjournals.org/ Downloaded from gene. AChE catalyses the hydrolysis of the neurotransmitter acetylcholine (ACh) and is crucial for cholinergic neurotransmission (Soreq and Seidman, 2001) . AChE is a target of a plethora of toxins including snake venom, insecticides and chemical weapons (Soreq and Seidman, 2001 ). The pharmacological use of AChE inhibitors includes treatment of the autoimmune disease myasthenia gravis, glaucoma and Alzheimer's disease (Soreq and Seidman, 2001 ).
We have previously identified a recessive mutation in the ache gene of zebrafish (Behra et al., 2002) . Zebrafish AChE is highly related to that of mammals (Bertrand et al., 2001) . A point mutation leading to replacement of a conserved serine at position 226 by an asparagine abolishes AChE enzymatic activity in homozygous mutant embryos. Mutants have impaired motility and develop a severe myopathy during the second day postfertilisation (Behra et al., 2002) . The myopathy is due to an over-activation of the muscle by accumulating ACh; genetic impairment of the nicotinic acetylcholine receptor (nAChR) on the muscle cell prevents the development of the myopathy in ache mutants (Behra et al., 2002) .
The zebrafish presents a unique situation among vertebrates as the AChE is the only ACh hydrolysing enzyme in this organism. Homozygous ache mutant embryos and larvae lack detectable ACh hydrolysing activity and heterozygous adults show 50% of the ACh cleaving activity of wildtype siblings (Behra et al., 2002) . Moreover, the zebrafish genome does not encode a functional butyrylcholine esterase, a related enzyme that can also hydrolyse Ach (Bertrand et al., 2001 ).
The phenotypic traits of the ache mutant provide criteria for a vertebrate that lacks ACh hydrolysing activity entirely and can thus serve as comparative standards for the specificity of AChE inhibitors. In this report, we used the zebrafish embryo as a whole organism model to test the specificity of chemicals. We chose four drugs known to be AChE inhibitors (Cousin et al., 1998; Giacobini, 1998) and compared their effects with the consequences of genetic elimination of AChE activity. Only one test compound, (galanthamine, (GAL)) reproduced the ache mutant phenotype. The other three compounds (eserine (ESE), tacrine (TAC) and edrophonium (EDRO)) showed effects in addition to those observed in ache mutants demonstrating secondary target effects. In general terms, this work illustrates the power of the combination of zebrafish genetics with toxicological testing in the verification of drug target specificity and demonstrates the potential of the zebrafish embryo in the assessment of the toxicity of chemicals. 
Experimental protocol Fish stocks and embryo production
The wildtype zebrafish line wt ABO is a cross between fish purchased from a pet shop and the AB strain (Eugene, Oregon) and was maintained as inbred line for several years in the lab. Fish were bred and raised as described (Westerfield, 1993) . The origin of the ache and the nic1 mutant and the -3.1ngn1:gfp transgenic zebrafish were described (Behra et al., 2002; Blader et al., 2003; Sepich et al., 1998 
Inhibitor treatment
Embryos were treated with the inhibitors (GAL (1,2,3,4,6,7,7a,11c-octahydro-9-methoxy-2methylbenxzofuro[4,3,2-efg] [2]benzazocin-6-ol), ESE (1'methylpyrrolidino (2':3':2:3) 1,3 dimethylindolin-5-yl N-methylcarbamate), TAC (1,2,3,4-tetrahydro -9-aminoacridine) and EDRO ((3-hydroxyphenyl) dimethylethylammonium bromide) diluted in 10% Hanks's solution (Westerfield, 1993) For video analysis, embryos or young larvae were embedded in low melting point agarose as described (Behra et al., 2002; Westerfield, 1993) . To allow recording of motility, the trunk and tail of embedded embryos was freed of agarose. Embryos were mounted under a dissecting microscope (Leica MZ FLIII) equipped with a CoolSnap video camera. Embryos were touched with a blunt metal rod at the body flank to trigger the startle response and swimming movements. Touch-evoked movements of embryos were monitored using the video camera recording 50 frames per second. Representative frames were overlaid to indicate the movement.
For measurement of the duration of swimming, individual embryos were placed in a 5 cm petridish. After touching the embryos, movement was recorded with the video camera.
The duration of the swimming movements was measured with a stop watch from the recorded film. In these experiments only the swimming movements were scored. However, most inhibitor-treated and ache mutant embryos exhibited the startle response entailing a very fast bend of the body axis in the 10 millisecond range followed by a slow straightening of the body axis over the next 200 milliseconds. This movement was not scored as swimming movement since, with the exception of the initial bend, it does most likely not reflect an active movement. The data sets were subjected to statistical analysis with the Student's t-test to assess the significance of the similarities and differences between wildtype, ache mutant and inhibitor treated embryos.
Measurement of AChE enzymatic activity
Detection of AChE activity in situ was adapted from Karnovsky (Karnovsky and Roots, 1964) . For IC 50 measurements, AChE activity in supernatants of 4 day old larvae was carried out in triplicate as described (Behra et al., 2002; Bertrand et al., 2001; Ellman et al., 1961) . Larvae were killed by incubation on ice for 5 min. Extract was prepared by passing embryos ten times through a 26-gauge syringe in LST buffer (20 mM Tris/HCl pH 7.0, 5 mM EDTA, 1% Triton X-100). Afterwards extracts were centrifuged 1 min at 10000 rpm and supernatants were diluted in 100 mM phosphate buffer pH 7.0, 0.5 mM DTNB and preincubated with varying concentrations of inhibitors for 10 min (TAC, GAL, EDRO) or 60 min (ESE). Acetylthiocholine was added to a final concentration of 1 mM before real-time determination of acetylthiocholine hydrolysis at 412 nm (Ellman et al., 1961) . IC 50 were deduced from the plots of activity versus inhibitor concentration from three independent measurements at every inhibitor concentration.
Measurement of AChE activity in 2 day old ESE-treated embryos was carried essentially as described above except that inhibitor was not added to the extract.
Confocal microscopy and birefringence
Transgenic embryos were mounted in Aqua Polymount and were analysed in lateral views with a confocal microscope. Optical sections were taken through the entire embryo.
Stacks of images were used in 3D reconstruction, to generate lateral and transverse projections. Image processing was carried out with the programs tcstk and timt (Behra et al., 2002) , further details and the programs are available from Jean-Luc Vonesch (xryan@example.net).
Birefringence was analysed as previously described (Behra et al., 2002) . Briefly embryos were embedded in methylcellulose (Westerfield, 1993) , mounted under a dissecting microscope (Leica MZ FLIII) and illuminated with polarised light. The parallel alignment of normal muscle fibres reflects polarised light. The degree of reflection was translated into a colour code to reveal the differences in intensities between wildtype, mutant and inhibitor treated embryos.
Microinjection
Microinjection of 10 -2 M GAL into the yolk of one cell stage embryos was carried out with a pressure driven microinjector (Eppendorf) as described (Westerfield, 1993) . The injection volume was measured by injection into an oil droplet on an electron microscope grid and the volume of a zebrafish embryo was estimated to be approximately 1000 nl.
Application of penetration enhancers
In experiments, which were aimed to test the function of penetration enhancers, GAL was applied alone or together with the penetration enhancers from onset of gastrulation (50% epiboly; see (Westerfield, 1993) for staging). In pilot experiments, the range of concentration of each penetration enhancer, which did not show toxicity, was tested. Dimethylsulfoxide applied at 2% (v/v) did not reduce the effective concentration of GAL and caused toxic effects when administered alone at 5% (v/v). Caprate (0.005%), hexadecyltrimethylammonium bromide and cetyltrimethylammonium bromide gave a very weak enhancement of the GAL effect or were ineffective.
Results

Inhibitors of AChE phenocopy the motility defect of the ache mutation.
To validate a use of zebrafish embryos as model for testing the specificity of AChE inhibitors, we chose 4 compounds known to be inhibitors of AChE. These include the drugs ESE, GAL, TAC and EDRO (chemical names see Material and Methods, (Cousin et al., 1998 and references therein).
We first assessed the effect of the compounds on motility of the embryo and young larva. Wildtype embryos show first signs of motility at 18 hours post-fertilisation (hpf). This initial beating of the tail is spontaneous, but becomes touch-evoked by about 36 hpf. Motility in response to touch has two phases: an immediate strong bend away from the source of stimulation, the so-called startle response, followed by a longer phase of swimming movements that varies in duration. Starting from 27 hpf, spasmodic contractions of the body axis can be observed in ache mutants while wildtype embryos perform smooth, wave-like movements of the trunk and tail (Behra et al., 2002) . By 48 to 72 hpf, motility of the mutants is strongly impaired (Fig. 1A, B) . We determined the concentration of each of the four compounds, at which they affect motility in a similar manner as the ache mutation (Fig. 1, Tab. 1). Whole embryos were exposed to the compounds from the 5-somite stage onwards, the time, at which ache starts to be expressed (Bertrand et al., 2001) . Embryos bathed in 10 -4 M ESE, 10 -2 M EDRO, 10 -3 M GAL or 10 -5 M TAC showed reduced motility at 27 and 48 hpf in a manner similar to ache mutants (Fig. 1C, Fig. 2 , Tab. 1). The embryos under all treatment conditions had beating hearts, indicating that impaired motility is not due to a lethal effect of the compounds. Increasing the concentration by tenfold did not elicit a stronger inhibition of motility and caused unspecific effects in the case of EDRO and TAC such as necrosis in the brain (data not shown).
To quantify the impairment of motility, we measured the duration of swimming movements after touching wildtype, ache mutant or inhibitor-treated embryos (Fig. 2 , Tab. 1). Wildtype embryos carried out swimming movements for an average of 2.8 seconds after being stimulated. In contrast, ache mutants showed on average only a very short phase of swimming. Both GAL and ESE reduced the average duration of swimming by 95% (Fig. 2) while TAC and EDRO were slightly less effective in reducing the average swimming episode (86% and 70%, respectively (Fig. 2, Tab. 2)). The data sets were pairwise subjected to the Student's t-test. In comparison to that of wildtype embryos, the duration of swimming of ache mutant and inhibitor treated embryos was significantly reduced (p<<0.05, Tab. 2). The difference between ache mutant, ESE and GAL treated embryos is not significant (p>0.05, Tab 2). The lower efficiency of TAC and EDRO in reducing the duration of swimming is, however, significant (p<0,05, Tab. 2). Although ache mutant and inhibitor-treated embryos show strong reductions in swimming duration, they responded in many instances with one fast bend of the body axis. Moreover, the inhibitor treated embryos, like ache mutants, had beating hearts demonstrating that impaired motility is not due to a general toxic effect of the drugs (Tab. 2). Thus, zebrafish embryos treated with inhibitors of AChE have impaired motility similar to that of embryos that carry a loss-of-function mutation in the ache gene.
Inhibitors differ in their ability to induce muscle degeneration
Lack of AChE activity causes a progressive myopathy in homozygous ache mutant embryos. Mutant muscle fibers form vacuoles and myofibrils are not aligned in parallel as in wildtype embryos (Behra et al., 2002) . As a consequence, the reflection of polarised light (or birefringence) from the flank of mutant embryos is severely reduced at 48-72 hpf (compare Fig. 3A , B). We tested whether the AChE inhibitors would cause similar muscle defects.
Embryos were exposed to the inhibitors from the 5-somite stage and were analysed under by guest on January 11, 2016
http://toxsci.oxfordjournals.org/ Downloaded from polarised light at 72 hpf. GAL caused severe muscle defects in wildtype embryos (Fig. 3D) that were not increased by absence of the ache gene (Fig. 3E) . Development of the myopathy in ache mutants requires the nicotinic acetylcholine receptor (nAChR), since mutations in the 1 subunit of nAChR (nic1) suppress the myopathy of ache mutants (Fig. 3J to L and (Behra et al., 2002) for details). In contrast to wildtype embryos, GAL-treated, nic1 mutant embryos that lack functional nAChR (Sepich et al., 1998) do not develop a myopathy ( Fig. 3F compared with C, D). Thus, the GAL-induced muscle degeneration depends on a functional nAChR similar to the myopathy caused by lack of ache gene activity. In comparison to GAL (Fig. 3D ), EDRO and TAC induced a weaker reduction in birefringence (data not shown) in agreement with their slightly weaker effect on motility (Fig. 2 , Table 2 ).
Curiously, ESE, even though it impaired motility strongly ( Fig. 2) , did not cause a significant reduction in birefringence (Fig. 3G) . ESE may not inhibit AChE at all in the zebrafish or may have additional activities. To exclude the first possibility, we performed histochemical and spectrophotometric measurements of AChE activity in extracts or intact embryos treated with ESE. Embryos exposed to 10 -5 ESE showed a reduction of AChE histochemical activity and exposure to 10 -4 M ESE almost completely abolished the histochemical reaction (Fig. 4A to D) comparable to that seen in the ache mutant (Fig. 4D) .
Similarly, when measured by the colorimetric Elman assay, the AChE activity was reduced by 72% and 91% in extracts from embryos treated with 10 -5 and 10 -4 M ESE, respectively.
Hence, ESE is able to inhibit zebrafish AChE efficiently. The lack of a myopathy is thus likely due to a secondary target effect of ESE. The lack of the myopathy in embryos that lack a functional nAChR renders the nAChR a possible secondary target of ESE. If this notion is correct, the myopathy of ache mutant embryos should be suppressed by treatment with ESE.
Indeed, treatment of ache mutants with ESE almost completely abolished the myopathy (Fig.   3H ) observed in untreated ache mutants (Fig. 3B) . These results show that ESE has a secondary effect. This is most likely caused by antagonistic interaction with the nAChR. In vitro studies suggest that ESE can act as an antagonist of the Torpedo nAChR (Kawai et al., 1999) .
Secondary effects of inhibitors in the nervous system
To assess a possible effect of the AChE inhibitors on the developing nervous system, we utilised a transgenic line that expresses the green fluorescent protein (gfp) gene under control of the regulatory sequences of the zebrafish neurogenin1 gene (-3.1ngn1:gfp) (Blader et al., 2003) . The neurogenin1 gene is a neural determination gene, which controls differentiation of Rohon Beard sensory neurons involved in the perception of touch (Korzh and Strahle, 2002) . The transgene is strongly expressed in Rohon Beard neurons (Fig. 5A ).
In addition, a few scattered interneurons located immediately below the dorsal longitudinal fascicle (dlf) are marked by weak GFP expression (Fig. 5A) . The overall pattern of GFP expression is unchanged in ache -/-embryos (n=20) at 27 hpf ( Fig. 5B) suggesting that lack of ache activity does not affect the expression of the transgene and the pattern of neurogenesis at this stage. When embryos were exposed to either EDRO (Fig. 5C ) or TAC (Fig. 5D ) from the 5-somite stage to 27 hpf, the pattern of GFP-expressing neurons was abnormal (69% treated embryos, n=19; 70% treated embryos, n=37, respectively). Ectopic, GFP-expressing neurons were noted at the ventral aspects of the neural tube in the drug-treated embryos suggesting that the two drugs caused aberrant neurogenesis or cell migration (Fig. 5C, D ).
An abnormal pattern of neurogenesis was not observed in the ache mutants ( Fig. 5B; n=20 ).
This effect is not the result of inhibition of AChE activity and is likely to reflect a secondary target effect.
These drastic changes in neuronal pattern were also not observed in embryos treated with ESE or GAL (Fig. 5F , E, n=35 and n=33, respectively). Occasionally, ESE or GAL-treated embryos were noted that had slightly displaced cells (Fig. 5F , E, arrow, 23% and 21%, respectively). However, these cells were also observed in untreated embryos at a similar frequency.
The inhibitors block zebrafish AChE effectively at micro-to nanomolar ranges.
When assessed by their dose-dependent effect on motility (Fig.1 , Tab. 1) or by in situ analysis of AChE activity in the case of ESE (Fig. 4B,C) , the effective blocking concentration of each of the 4 inhibitors is high in comparison to reported IC 50 values of AChEs from other species (Ariel et al., 1998; Harvey 1995; Shafferman et al., 1992; Stein and Lewis, 1969) . Three reasons could account for this: first, the inhibitors have lower affinities to the AChE of zebrafish than to that of other vertebrates. Second, the zebrafish embryo may take up these chemicals from the environment rather inefficiently hence requiring higher doses of inhibitor. Finally, one could also envisage that the compounds are metabolised in the embryo.
To assess the first possibility, the IC 50 of the four inhibitors was determined in extracts of zebrafish larvae. The extracts were pre-incubated with varying concentrations of the inhibitors before AChE activity was measured spectrophotometrically (Ellman et al., 1961) .
ESE and TAC inhibited zebrafish AChE half-maximally at 125 +/-15 nM and 114 +/-23 nM, respectively (Fig. 6) . The respective IC 50 values of GAL and EDRO were 2800 +/-21 M and 1300 +/-100 nM. These IC 50 values are in the range of those reported for AChEs of other species (Ariel et al., 1998; Harvey, 1995; Shafferman et al., 1992; Stein and Lewis, 1969) .
Thus, the zebrafish AChE is as sensitive to the inhibitors as those of other vertebrates.
To exclude the possibility that the drugs are metabolised in the embryo, GAL was injected into the yolk cell of one-cell stage embryos. We injected approximately 1 nl of 10 -2 M GAL per embryo resulting in an intraembryonic concentration of about 10 -5 M assuming free diffusion within the embryo and no reduction due to growth of the embryo and diffusion out of the embryo until the 72 h-stage. All injected embryos (n=50) showed impaired motility and reduced reflection of polarised light at 72 hpf in comparison to mock injected controls (Fig. 7A, B) . Assuming no losses due to dilution or diffusion between the injection and examination of the effect 72 h later, the estimated intra-embryonic concentration is at least 10-fold lower than that required in bath-application to "chemocopy" the ache mutant phenotype.
Thus, intra-embryonic degradation of the drugs is unlikely to account for the relative insensitivity of whole embryos.
Taken together, these results suggest that GAL penetrates the zebrafish embryo ineffectively. As microinjection is not the most convenient way to deliver compounds to a large number of embryos, we next assessed whether the drug effect could be improved by coapplying penetration enhancers. Thus, impaired penetration is a likely explanation for the relative insensitivity of whole embryos and the barrier can be overcome by applying the penetration enhancer Tween 80.
Discussion
We assessed here the potential of wildtype and mutant zebrafish embryos as model for the specificity of inhibitors of AChE. Using this class of inhibitors, this work was also aimed at evaluating in general the use of wildtype and mutant zebrafish embryos in testing the toxic or teratological effect of chemicals. Among the four compounds tested, only GAL "chemocopied" the ache mutant phenotype. Like genetic loss of ache function, GAL treatment impaired motility, caused a myopathy, but did not affect the differentiation of neurons in the spinal cord. Moreover, the myopathy in ache mutants (Behra et al., 2002) and GAL treated embryos (this study) require both an intact nAChR. The other inhibitors had additional effects not seen in ache mutants. Both TAC and EDRO caused impaired neuronal development that was not detected in ache mutants. ESE even cured the muscle defects of the ache mutants. As a similar suppression of the muscle phenotype of ache mutants is observed, when the AChR is genetically removed (Behra et al., 2002) , this suggests that ESE acts as an antagonist of the AChR. This notion is in agreement with previous in vitro studies (Kawai et al., 1999) , in which it was shown that ESE acts as an antagonist of the nAChR. In summary, our work demonstrates the power of wildtype and mutant zebrafish embryos to assess the target specificity of AChE inhibitors.
We noticed also a drawback in that the concentrations required to "chemocopy" the ache mutant phenotype in intact embryos are higher in comparison to the reported in vitro inhibitory concentration for AChEs from other species (Ariel et al., 1998; Harvey, 1995; Shafferman et al., 1992; Stein and Lewis, 1969 ). This does not appear to be due to a low affinity of the inhibitors to zebrafish AChE or degradation of the compounds in the embryos. present an obstacle for second phase toxicological assessment of candidates or the analysis of the toxicological mechanism. Interestingly, not all penetration enhancers were equally effective, suggesting that the type of penetration enhancer may have to be taken into consideration for each compound class to be tested.
The effective in vivo dose to impair motility appears to be linked to the chemical structure of the AChE inhibitors. EDRO is the least efficient (10 -2 M) to inhibit motility in bath applications even though its in vitro IC 50 is in the m range. ESE "chemocopies" the ache mutant phenotype less effectively than TAC, but both compounds inhibit the AChE activity with an IC 50 around 100nM. Other compounds were previously applied to zebrafish embryos at micro-and nanomolar concentrations and caused significant effects (Peterson et al. 2000; Peterson et al., 2001) . We show that, at least in the case of GAL, direct injection of the compound or the co-administration of the penetration enhancer Tween 80 can reduce the effective dose in the embryos suggesting that the up-take of the AChE inhibitors may represent a barrier for the AChE inhibitors.
Our work illustrates the potential of zebrafish in assessment of toxicological and teratological effect of compounds in that zebrafish mutants and mutant phenotypes act as blueprints of drug effect to assess target specificity and to gain insight into the toxic mechanism in a whole organism set-up. Ideally, if an inhibitor is specific it should reproduce the phenotype of a loss-of-function mutation faithfully with no additional effects. However, reality can be more complex than this theoretical concept. Complications may be imposed by the complexity of the responses of intact organisms. First, the phenotypes of a loss-offunction mutation may not reflect the effect of an inhibitor that blocks its target only partially as sub-maximal inhibition may have different phenotypic consequences. Second, detoxifying pathways may break down a chemical to metabolites that act unspecifically, even though the administered inhibitor is perfectly specific for its target. Another complication may arise by the fact that genetically altered organisms can have varying phenotypes depending on the specific lesion and the genetic background. We have not observed variation of the ache mutant phenotypes utilised here, even when crossed into distantly related strains such as the WIK mapping strain (MB and US, unpubl.) . However, this does not mean that this may not present a problem in other genetic backgrounds and for other genetic lesions in the zebrafish.
Despite these caveats, we believe that, with the increasing number of cloned mutant loci, the zebrafish will provide a simple cost-effective platform to assess the effects of chemicals on many developmental and physiological processes. The potential of the zebrafish is even further increased by the steadily growing number of transgenic lines that permit monitoring of many physiological processes in the living animal. The small size, transparency and extra-uterine development of the zebrafish embryo allow the application of simple assays, which can easily be modified to satisfy medium-and possibly also highthroughput applications. In summary, even though the concentrations required to achieve an effect may be relatively high for certain chemicals, the zebrafish embryo can provide a valuable whole-organism-assay to satisfy the demand for efficient toxicological test systems by regulators and industry. Efficient inhibition of zebrafish AChE by GAL, ESE, TAC and EDRO in vitro
Legends
Extracts from 4 day old embryos were incubated with increasing concentrations of ESE, GAL, EDRO and TAC and AChE activity was measured using the Ellman assay. IC 50 are in the nano-to micromolar range. 

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