* Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, France;
Forschungszentrum Karlsruhe, Karlsruhe, Germany; and
Unite Différenciation Cellulaire et Croissance INRA, Montpellier, France
Received August 8, 2003; accepted October 20, 2003
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ABSTRACT |
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Key Words: zebrafish; acetylcholinesterase; inhibitors; animal model; secondary drug targets.
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INTRODUCTION |
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Besides associated ethical concerns, the use of mammals experimentally is expensive and labor 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, thereby allowing systematic compound screens (Peterson et al., 2000) from earliest life stages onwards. Moreover, a large number of mutations affecting signaling 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
).
For this study, we investigated the application of wild-type and mutant zebrafish embryos in drug testing by phenotypic comparisons with a mutation in the acetylcholinesterase (ache) gene. AChE catalyzes the hydrolysis of the neurotransmitter acetylcholine (ACh) and is crucial for cholinergic neurotransmission (Soreq and Seidman, 2001). AChE is the target of many 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 Alzheimers disease (Soreq and Seidman, 2001
).
We have previously identified a recessive mutation in the ache gene of zebrafish (Behra et al., 2002). Zebra fish 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 postfertilization (Behra et al., 2002
). The myopathy is due to an overactivation 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-hydrolyzing enzyme in this organism. Homozygous ache mutant embryos and larvae lack detectable ACh-hydrolyzing activity and heterozygous adults show 50% of the ACh cleaving activity of wild-type siblings (Behra et al., 2002). Moreover, the zebrafish genome does not encode a functional butyrylcholine esterase, a related enzyme that can also hydrolyze ACh (Bertrand et al., 2001
).
The phenotypic traits of the ache mutant provide criteria for a vertebrate that lacks ACh-hydrolyzing 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 chemical toxicity.
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MATERIALS AND METHODS |
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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 solution (Westerfield, 1993) from the 5-somite stage onwards. Solutions were replaced each day. Unless stated otherwise, 10-4 M ESE, 10-3 M GAL, 10-2 M EDRO, and 10-5 M TAC were used. These concentrations were determined in motility assays and by morphological inspection of embryos to phenocopy the ache mutant phenotype with respect to motility most closely. Higher concentrations of TAC and EDRO caused necrosis in the neural tube and were thus regarded to have a general toxic effect on the embryos.
Motility assays.
For video analysis, embryos or young larvae were embedded in low melting point agarose, as described previously (Behra et al., 2002; Westerfield, 1993
). To allow recording of motility, the trunks and tails of embedded embryos were freed of agarose. Embryos were mounted under a dissecting microscope (Leica MZ FLIII, Leica, Bensheim, Germany) equipped with a CoolSnap video camera (Roper Scientific, Munich, Germany). 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/s. Representative frames were overlaid to indicate the movement.
For measurement of the duration of swimming, individual embryos were placed in a 5-cm petri dish. After touching the embryos, movement was recorded with the video camera. The duration of the swimming movements was measured with a stopwatch 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 with a very fast bend of the body axis in the 10 ms range followed by a slow straightening of the body axis over the next 200 ms. 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 Students t-test to assess the significance of the similarities and differences among wild-type, 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 IC50 measurements, AChE activity in supernatants of 4-day-old larvae was carried out in triplicate, as described previously (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 for 1 min at 10,000 rpm; supernatants were diluted in 100 mM phosphate buffer, pH 7.0, and 0.5 mM DTNB and preincubated with varying concentrations of inhibitors for 10 min (TAC, GAL, and 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
). IC50 values 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 (Polysciences, Warrington, PA) and were analyzed in lateral views with a confocal microscope (Leica, Bensheim, Germany). 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 are available from Jean-Luc Vonesch (jlv{at}igbmc.u-strasbg.fr).
Birefringence was analyzed as described previously (Behra et al., 2002). Briefly, embryos were embedded in methylcellulose (Westerfield, 1993
), mounted under a dissecting microscope (Leica MZ FLIII), and illuminated with polarized light. The parallel alignment of normal muscle fibers reflects polarized light. The degree of reflection was translated into a color code to reveal the differences in intensities among wild-type, 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 AG, Hamburg, Germany), as described previously (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 that were intended to test the function of penetration enhancers, GAL was applied alone or together with the penetration enhancers from the 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.
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RESULTS |
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We first assessed the effect of the compounds on motility of the embryo and young larva. Wild-type embryos show the first signs of motility at 18 h postfertilization (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 could be observed in ache mutants, while wild-type embryos performed smooth, wave-like movements of the trunk and tail (Behra et al., 2002). By 4872 hpf, motility of the mutants was strongly impaired (Fig. 1A and B
).
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The Inhibitors Block Zebrafish AChE Effectively at Micro- to Nanomolar Ranges
When assessed by their dose-dependent effect on motility (Fig. 1 and Table 1
) or by in situ analysis of AChE activity in the case of ESE (Fig. 4B and C
), the effective blocking concentration of each of the four inhibitors was high in comparison to reported IC50 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: (1) the inhibitors have lower affinities to the AChE of zebrafish than to that of other vertebrates; (2) the zebrafish embryo may take up these chemicals from the environment rather inefficiently, hence requiring higher doses of inhibitor; and/or (3) the compounds are metabolized in the embryo.
To assess the first possibility, the IC50 of the four inhibitors was determined in extracts of zebrafish larvae. The extracts were preincubated 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 and 114 ± 23 nM, respectively (Fig. 6
). The respective IC50 values of GAL and EDRO were 2800 ± 21 µM and 1300 ± 100 nM. These IC50 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 the AChE of other vertebrates.
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DISCUSSION |
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We noticed a drawback in that the concentrations required to "chemocopy" the ache mutant phenotype in intact embryos were 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. A likely explanation is the inefficient uptake of the compounds. While this relative insensitivity of zebrafish embryos may be a hindrance in primary screening programs of compound libraries where the available amounts of chemical are rather limited, it may not 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 was the least efficient (10-2 M) at inhibiting motility in bath applications even though its in vitro IC50 is in the micromolar range. ESE "chemocopied" the ache mutant phenotype less effectively than TAC, but both compounds inhibited the AChE activity with an IC50 around 100 nM. Other compounds were previously applied to zebrafish embryos at micro- and nanomolar concentrations and caused significant effects (Peterson et al., 2000, 2001
). We show that, at least in the case of GAL, direct injection of the compound or the coadministration of the penetration enhancer Tween 80 can reduce the effective dose in the embryos, suggesting that the uptake of the AChE inhibitors may represent a barrier for the AChE inhibitors.
Our work illustrates the potential of zebrafish in the assessment of toxicological and teratological effects of compounds; zebrafish mutants and mutant phenotypes act as blueprints of drug effect for assessing target specificity and gaining insight into the toxic mechanism in a whole organism setup. 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. Firstly, the phenotypes of a loss-of-function mutation may not reflect the effect of an inhibitor that blocks its target only partially, as submaximal inhibition may have different phenotypic consequences. Secondly, 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 from 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 used here, even when crossed into distantly related strains such as the WIK mapping strain (M.B. and U.S., unpublished). 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 extrauterine development of the zebrafish embryo allow the application of simple assays, which can easily be modified to satisfy medium- and possibly also high-throughput 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.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed at Institut de Genetique et de Biologie Moleculaire et Cellulaire, 1 rue Laurent Fries, BP10142, 67404 Illkirch, France. Fax: +33 3 88 65 32 01. E-mail: uwe{at}igbmc.u-strasbg.fr.
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