Zebrafish:a new model on the pharmaceutical catwalk
Ulrike Langheinrich
Summary
Zebrafish is recognized as one of the most important vertebrate model organisms;however,its value in pharm-acological studies has not been extensively explored and exploited.In this review,I summarize significant findings about the effects of drugs and medicines on important physiological process in zebrafish.Our experiments have shown that cardiovascular,anti-angiogenic and anti-cancer drugs elicit comparable respons in zebra-fish embryos to tho in mammalian systems.Similar obrvations have been reported by other laboratories,exposing zebrafish to a variety of pharmaceutical active compounds affecting a range of different process.All the data summarized indicate that zebrafish reprents a very valuable organism for different kinds of pharmaco-logical studies,such as screenings of chemical libraries,lead validation and optimization,mode-of-action studies,analysis of gene function,predictive toxicology and teratogenicity,pharmacogenomics and toxicogenomics.Zebrafish pharmacological assays have specific advan-tages compared to in vitro cell culture studies and in vivo experiments using mice,complementing the assays to give valuable guides for future tests of new drugs for human therapy.BioEssays 25:904–912,2003.ß2003Wiley Periodicals,Inc.
Introductionlovethewayyoulie
The teleost zebrafish (Danio rerio )is a small tropical fish (adults are about one inch long),which produces optically transparent embryos that develop rapidly outside the mother’s body .By 6days postfertilization (dpf),a complex circulatory system and counterparts of most mammalian organs have developed.Traditionally ,zebrafish has been mainly ud as a model organism in the fields of molecular genetics and deve-lopmental biology of vertebrates.More recently ,however ,its value as a model organism for drug target discovery ,target validation,drug-finding strategies and toxicological studies has begun to be recognized.Although zebrafish proteins display less than 70%identity to their human orthologues,conrvation in the functional domains,such as substrate-binding regions (often the drug-binding targets),is consider-ably higher ,approaching values of 100%similarity .This is the structural basis for the obrvation that a number of drugs elicit comparable effects in zebrafish and in humans.Applica-tion of drugs to zebrafish is simple as embryos rapidly absorb low molecular weight compounds,diluted in the surrounding media,through skin and gills.Highly hydrophilic compounds must be injected at the 1-to 4-cell stage into the yolk sac or later into the sinus venosus or vein.Older animals,from 7dpf to the adult stage,absorb compounds orally from the tank water .Fig.1shows schematically a possible experimental flow for drug experiments using zebrafish embryos.
Pharmacologically induced knockdown of protein function can complement or even replace antin approaches and could help to characterize mutant phenotypes and the function of specific genes in zebrafish.In addition,new drugs might be identified by screening large numbers of zebrafish embryos for specific effects following exposure to a library of low molecular weightcompounds.Whole-animal high-throughput screenings have been so far restricted to invertebrate model organisms,like the fruitfly Drosophila melanogaster (1)and the nematode Caenorhabditis elegans .(2)The tough cuticle,however ,pre-vents many compounds from entering the organisms;fur-thermore,proteins specific to vertebrate organisms will obviously not be targeted.Early in vivo hit validation and optimization using zebrafish embryos is another field where zebrafish has certain advantages compared to cell culture systems and in vivo assays in mice.
In this review,I will summarize the current available data on the effects of drugs on important physiological process in zebrafish (angiogenesis,hemostasis,apoptosis and prolifera-tion,lipid metabolism,inflammation,neural tolerance)and zebrafish’s particular value for a number of different pharma-cological approaches will be discusd.One aim of this review is to encourage both rearchers and the pharmaceutical industry to exploit the similarity of drug-binding regions between human and zebrafish proteins for the development of new and better human therapeutics.
Angiogenesis
In recent years,understanding how vesls are formed has attracted considerable interest.This is mainly due to the hope that,in future,a broad range of different,genetically hetero-geneous cancer types can be treated by inhibiting a common process occurring in most tumors,namely the formation of new vesls from existing ones (angiogenesis)and thereby
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BioEssays 25:904–912,ß2003Wiley Periodicals,Inc.
Ulrike Langheinrich,Exelixis Deutschland GmbH,Spemannstras
35,D-72076Tu
¨bingen,Germany .E-mail:U. DOI 10.1002/bies.10326
Published online in Wiley InterScience (www.interscience.wiley .com).
商务关系disrupting the nutrient flow to the cancer cells.The optical transparency and its ability to survive witho
ut functioning circulation for 3–4days make the zebrafish especially amenable to in vivo vascular cell biology .(3)In contrast,in
mammals,most vascular cells are deep within the animal and not accessible for optical imaging.The vascular system of zebrafish larvae can be imaged through fluorescence micro-scopy ,using transgenic animals or by injecting fluorescent dextran beads into the vascular system (microangiography),or through an enzymatic assay ,exploiting the vascular localization of alkaline phosphata in young zebrafish.(4)A number of genes involved in vertebrate ,vascular endothelial growth factors,angiopoietins,ephrins and their respective receptors,have been identified in zebra-fish and been shown to have similar functions to tho in mammals.(3,4)
Knockdown of VEGF ,for example,induced by injecting antin oligonucleotide morpholinos,targeting VEGF ,into zebrafish embryos (5)leads to a complete lack of all vesls.A phenocopy can be obtained by bathing embryos in medium containing PTK787/ZK222584(an anilinophthalazine com-pound),suppodly lective for VEGF receptors (VEGFR).(6)The specificity of the compound has been shown by the lack of deleterious effects on the general morphology and by a significant reduction of endothelial cells in the region of the dorsal artery and posterior cardinal
vein at 24hpf.When the compound was administered relatively late,only the interg-mental vesls failed to form and washout of the drug induced a slight recovery .(6)The data indicate a major advantage of drugs in comparison to morpholinos,the former can be added at any stage during embryo development and induction of chemotypes might be reversible.The high similarity of the human and zebrafish VEGFR tyrosine kina domain is the likely structural basis for the obrved specific effects of the human VEGFR inhibitors on zebrafish embryos.Screen-ings for new anti-angiogenic drugs appears feasible in zebrafish and,for example,Phylonix Inc.(Cambridge,MA;),a young company ,offers high-throughput compound screens (HTS)using zebrafish embryos with the aim of identifying new anti-angiogenic compounds.Embryos are put into 96-well plates,bathed with compounds and,after a few days of development,a fluorescent readout for endothelial alkaline phosphata activity is ud as an indicator for pro-and anti-angiogenic activity of the compounds tested.Hemostasis
儿童动画片排行榜Conrvation of the major hemostatic pathways involved in platelet function and coagulation,as well as the prence of both extrinsic and intrinsic pathways of coagulation,suggest that the zebrafish is also a relevant model for studying hemostasis and thrombosis.(7–9)Well-known human anti-coagulants,aspirin and warfarin,were added to the tank water of adult fish and specific diagnostic as
says,like adhesion/aggregation and ATP-relea assays,were performed on collected blood of drug-expod fish.Blood from aspirin-expod adult zebrafish demonstrated inhibition of arachido-nic acid-induced aggregation and agonist-induced
我想见你 英文ATP
Figure 1.Schematic diagram showing a possible experi-mental flow for zebrafish pharmacological assays.Embryos are collected from mating pairs and afterwards cultivated with embryo medium in Petri dishes or multiwell plates.At specific time points,drugs are added from stock solutions and the embryos further incubated for a certain duration.To avoid pigment formation,phenylthiourea (200m M)is included in the embryo medium.Various kinds of assays can then be ud in order to screen the larvae for specific effects.Except from readouts on live larvae,larvae can be fixed with paraformalde-hyde and afterwards stained for specific structures,proteins,metabolites etc.The examples show stainings for bone,lipids,primary motoneurons,cardiac and skeletal muscles,apoptotic cells,vascular system and blood.All stainings shown can also be performed in 96-well plates,each well containing up to 20larvae.The bottom of the wells is formed by a net,to simplify exchange of the bathing solutions.Staining procedures include fixation of the embryos in 4%paraformaldehyde,wash steps and bathing in methanol.Afterwards,depending on the assay ,veral additional steps,exchanges of the respective bathing solutions,are necessary for specific stainings.Screening of the chemotypes can either be done manually or automatically .
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relea,consistent with a partial dependence on the cyclooxy-gena pathway.(9)Since zebrafish hemostatic proteins,like their mammalian orthologues,depend on vitamin K-depen-dent g-carboxylation,warfarin,an inhibitor of this process,also had strong anti-thrombotic effects.Warfarin,added to the tank water,induced spontaneous visible bleeding in adult zebrafish and prolongation of modified prothrombin and partial throm-boplastin times,which are common diagnostic markers for coagulation disorders.Similar effects have been obtained with Protac,a human protein C(anticoagulant zymogen)activator, showing a remarkable conrvation of the drug-binding sites in zebrafish and human hemostatic proteins.(10)重庆礼仪
Heart function and circulation
Only a few studies have been published showing effects of cardiovascular drugs on zebrafish embryos.This is surprising as the two-chambered heart of zebrafish beats synchronously and regularly by24hpf and is easily accessible for drugs and microscopic obrvations,becau it lies just beneath the skin.
Vasoactive drugs,known to lower blood pressure in mammals,were applied to zebrafish embryos at5–6dpf and the vascular diameter determined,using a special technique called digital motion analy
sis.Application of the nitric oxide donor sodium nitroprusside resulted in a significant increa in both the venous and arterial vesl diameters of zebrafish embryos,whereas application of N(G)-nitro-L-arginine methyl ester(L-NAME),which competitively inhibits nitric oxide syntha,induced a significant decrea in the same dia-meter,similar to the effects on mammalian systems.Further-more,exposure to epinephrine,a beta1-adrenergic agonist, resulted in a strong increa in cardiac output and vesl diameter in larvae preincubated with L-NAME.Thus,impor-tant regulatory proteins of blood pressure are expresd from early on in zebrafish and are amenable to pharmacological studies.(10)
Recently,the zebrafish M(2)muscarinic acetylcholine receptor has been cloned and pharmacologically characteriz-ed.Its antagonistic affinity profile correlated well with the profile of human M(2)acetylcholine receptor.In zebrafish larvae,the agonistic drug carbachol inhibits the basal heart rate mediated by the M(2)receptor and,conquently,a knockdown of the M(2)receptor by injection of morpholinos, rendered carbachol ineffective.(11)The data are similar to mammals and promising.They show that new cardioactive human drugs and drugs eliciting side effects on the heart beat might be identified by bathing zebrafish in the respective compounds and scoring for heart beat abnormalities. Apoptosis and proliferation考研培训
panteraZebrafish has only been rarely ud as model organism for studying the basic process of cancer development.Knowl-edge of the occurrence and function of cancer-related genes is also quite limited in zebrafish.In the few published studies to date,rearchers attempted to induce tumor formation in zebrafish by exposing them to known cancerogenic com-pounds.Zebrafish embryos and fry,bathed for a short time (1–24hours)in a medium containing N-methyl-N0-nitro-nitrosoguanidine,when analyzed histologically6months later, were found to have developed a variety of different mench-ymal neoplasias.Hepatocellular adenoma was the most prevalent hepatic neoplasm.(12)In a recent paper,Langenau et al.(13)described transgenic zebrafish that expresd mou c-myc under the control of the zebrafish Rag2promotor.The fish developed T-cell acute lymphoblastic leukemia,thus providing a promising platform for genetic and drug screens aimed at finding drugs for treatment of c-myc-induced carcinogenesis.
The only study,to my knowledge,in which zebrafish embryos were treated with putative anti-cancer drugs,has been performed with the organo-metallic compound vanado-cene,a potent anti-proliferative agent that disrupts mitotic spindle formation in cancer cells.(14)This compound was microinjected into the cells of embryos in the2-cell stage and thereafter blocked cell division at the8-to16-cell stage of embryonic development followed by cell fusion and develop-mental arrest.In human cancer cells,proliferation is also inhibited by vanadocene,indicating a similar mode of action.
echo什么意思At Exelixis,Inc.and Exelixis Deutschland GmbH,one of our main goals is to discover new genes involved in specific steps during cancer development and metastasis formation and to develop drugs inhibiting the process.In zebrafish,we have studied the occurrence and function of two of the most intensively studied cancer-related genes:p53,a tumor sup-pressor and the oncogene Mdm2,p53’s main feedback regulator.(15)Both proteins can be successfully knocked down through an antin technology,introduced two years ago in zebrafish.It consists of blocking the translation of target proteins through the injections of antin oligonucleotide morpholinos(20–24mers)at the1-to2-cell stage.(5)Sum-marizing our data,p53knockdown did not cau a visible phenotype in unperturbed zebrafish embryos,whereas Mdm2 knockdown led to vere growth retardation,strong apoptosis and early death,induced by incread p53levels.A simultaneous knockdown of p53and Mdm2therefore com-pletely rescued the Mdm2phenotype,(15)as previously de-scribed for double knockout mice.(16,17)
We then asked whether the topoisomera inhibitor camptothecin,a widely ud anti-cancer drug,induces similar respons on a whole-organism level in the zebrafish as in mammalian systems.Relatively low concentrations of camp-tothecin(50–500nM)applied between24and30hpf,induced a strong increa in apoptosis as revealed by TUNEL staining for apop
totic cells.Cells in the brain,the eye and the spinal chord,most likely neurons,were predominantly labeled (Fig.2A).Morphologically,the embryos were retarded in development,displayed smaller head and eyes and darker
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tissues,especially in the eyes and brain.In contrast,if embryos deficient in p53,induced by injection of p53morpholinos,were expod to camptothecin,almost no increa in apoptosis could be detected (Fig.2B).The data suggest that the camptothecin-induced apoptosis in zebrafish resulted from an increa in p53activity ,probably due to DNA damage.In parallel to the increa in p53,we obrved very high levels mRNA levels of p21,one of the main downstream effectors of p53.(14)All the data are comparable to the effects of camptothecin in mammalian cell culture.(18)
Similar results have been obtained with the small molecule cyclin-dependent kina inhibitor roscovitine,which likely stabilizes and activates nuclear p53in tumor cells,possibly via a potent downregulation of Mdm2transcript and protein levels.(19)Buffer-injected control embryos,incubated for 24hours (24–48hpf)with 100m M roscovitine,became verely retarded and displayed dark tissues,whereas p53-deficient embryos,incubated with roscovitine,were only slightly retarded,showin
g slight heart edema and only the beginning of tail-tip necrosis (Fig.2C,D).The results suggest that roscovitine-induced growth retardation and apoptosis induc-tion in zebrafish were mainly mediated through an increa in p53activity ,similar to the human cell culture experiments.(19)
The experiments outlined above indicate that zebrafish embryos can be successfully ud in mode-of-action studies
and screenings for anti-cancer compounds.Combining pharmacological and genetic knockdowns (bathing of p53morpholino injected larvae with compounds),it can,for example,be determined whether the effects of apoptotic or anti-proliferative drugs are dependent on active p53.Many tumors lack functional p53,dramatically weakening the effects of most anti-cancer drugs;therefore,the favorite drugs would be tho that act independently of p53.New genes involved in proliferation or apoptosis might be identified in forward or rever genetic screens using anti-cancer drugs as nsitizing agents.
Lipid metabolism
During the first days of development,zebrafish embryos live on the lipids stored in their yolk sac.As the vascular system is established,the lipids are mobilized and transported to their target tissues;l
ater lipids can only be detected in the liver (Langheinrich et al.,unpublished data).Zebrafish has been shown to be a valuable model organism for studying mam-malian lipid metabolism:not only lipid processing through the intestine and hepatobiliary system but also respons to cholesterol blockers are similar in zebrafish compared to mammals.(20)In collaboration with Molecular Probes (Leiden,Belgium),Farber et al.developed fluorescently tagged or quenched phospholipids to assay lipid metabolism in living zebrafish.Larvae were soaked in PED6([N -((6-(2,4-dinitro-phenyl)amino)hexanoyl)-1-palmitoyl-2-BODIPY -FL-pentanoyl-sn -glycero-3-phosphoethanolamine),allowing subcellular visualization of phospholipa A 2activity due to cleavage of the substrate,resulting in unquenching.An inten staining of the gall bladder and subquently in the intestinal lumen was obrved.Gall bladder staining was completely blocked by atorvastatin,a potent 3-hydroxy-3-methylglutaryl coenzyme A reducta inhibitor ,which belongs to a substance class widely ud for lowering cholesterol in humans.Since exogenous bile reverd that effect in mice,the authors hypothesized that atorvastatin blocks the synthesis of cholesterol-derived biliary emulsifiers needed for dietary lipid absorption.(20)Using fluo-rescent reporter substrates,zebrafish HTS for drugs interfer-ing with lipid metabolism can be easily envisaged.
Inflammation
Studies on prostaglandin synthesis,a major step during the inflammation process,have thus far been mainly performed in mammals.Grosr ported studies on cyclooxygena-1and cyclooxygena-2in zebrafish,two enzymes catalyzing the conversion of arachidonate to prostaglandins.(21)Cycloox-ygena-1is expresd constitutively in mammals and is primarily responsible for prostaglandins regulating hemosta-sis,whereas cyclooxygena-2is an inducible enzyme,highly ,by growth factors,and is often overexpresd in many tumors.Recent papers indicate that cyclooxygena-2might also have an effect on angiogenesis.(22)
The
Figure 2.Induction of apoptosis and tissue degradation by camptothecin and roscovitine is dependent on p53.A:Zebrafish expod to 500nM camptothecin (24–30hpf)showed a strong increa in apoptosis,especially in the brain and spinal chord,as revealed by TUNEL staining.B:P53deficiency induced by injection of 3ng antin p53morpholinos at the 1-to 2-cell stage markedly suppresd the induction of apoptosis by camptothecin.C:Roscovitine,applied between 24and 48hpf (100m M)caud growth retardation and tissue degradation,especially in the brain and tail tip,p53deficiency strongly suppresd the effects.Scale bars:500m m.
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nonsteroidal anti-inflammatory drugs,acting non-specifically on both cyclooxygenas,and the specific cyclooxygena inhibitors are among the most widely ud products in the pharmaceutical industry.In order to characterize the function of both enzymes in zebrafish,both kinds of inhibitors have been added to the tank water of adult zebrafish and throm-bocyte aggregation,bleeding time and prostaglandin E2 formation analyzed later.
Indomethacin,a non-lective inhibitor,and NS-398,a specific cyclooxygena-2inhibitor,both inhibited prostaglan-din synthesis in adult zebrafish;IC50values were similar to the human enzymes.
The bleeding time was prolonged and thrombocyte aggregation reduced by exposure to indometha-cin.NS-398,in contrast,did not affect coagulation parameters. The results fit well with the obrvations in mammals (thrombocytes only express Cox-1),indicating that zebrafish might be amenable for screens aimed at the identification of new pharmacological compounds in inflammatory process. Further,important conclusions on the roles of both cycloox-ygenas in vertebrate development have been drawn from early knockdown of protein activity.Comparisons of human and zebrafish protein quences,especially in the drug-binding region,can lead to conclusions about the amino acids necessary for an effective inhibition by drugs.For example,two amino acids previously thought to be necessary for COX-2 inhibitor specificity,now appear to be less important,becau the zebrafish COX-2is different in the amino acids but shows a similar pharmacological profile to the human form. Drug abu and addiction
It appears quite attractive to include zebrafish in studies aimed at designing therapeutic strategies for the treatment of addiction.The effects of cocaine,nicotine and ethanol ex-posure on whole embryonic and adult zebrafish have been studied.(23–27)Darland and Dowling(23)showed that cocaine exposure has specific effects on zebrafish behavior and then performed a behavioral screen for cocaine-induced condi-tional-plate-preference in adult zebrafish.After a single exposure to cocain
e(10mg/l),85%of wild-type fish showed a positive change in preference and also a decrea in visual nsitivity.In the progeny of N-ethyl-nitrosourea-treated fish (ud to introduce point mutations),three families have been identified that had a high proportion of members showing an innsitivity to cocaine.In one family,dark adaptation and memory capacity were also altered,indicating that the mutations were localized in distinct genes that affect dopami-nergic signaling in the retina and brain.(23)
Nicotine,transiently applied to the medium bathing em-bryonic zebrafish,delays the development of spinal condary motoneurons.Furthermore,a long-lasting alteration in axonal pathfinding and long-term alteration in swimming behavior was obrved.The nicotinic receptor antagonist DH b E,an a4/ b2subtype-lective nAChR antagonist in mammals,blocked the action of nicotine completely,indicating the nAChRs play a significant role during zebrafish development.Thus,the mole-cular mechanisms and etiology of nicotine-induced neuronal toxicity can be studied using zebrafish embryos.(24) Zebrafish also show specific reproducible respons to ethanol.(25–27)Visual behavior and visual physiology was affected in zebrafish embryos expod to1.5%ethanol during distinct phas of eye development,the most-nsitive pha was between12and24hpf.Like children that have been expod to ethanol prenatally,zebrafish showed significant smaller eyes as
well as a reduction in visual acuity measured behaviorally.Furthermore,the waveform of the electroretino-grams differed between controls and ethanol-expod larvae and the visual thresholds were significantly higher than tho of controls.(25)When adult zebrafish were expod to ethanol, to study the chronic and acute effects of ethanol,(26)ethanol could be detected in the brain after a15minute exposure. Acute exposure to ethanol incread the average distance between the fish and inhibited the startle reaction.Chronic exposure induced tolerance in the long-fin-striped strain but not in the wild-type strain,indicating,as in rodents,that the genotype can influence the development of tolerance,which might involve adaptation of CNS neurons.(27)In summary, zebrafish might be an excellent model for studying addiction and also for the identification of drugs curing addiction. Toxicology and teratogenicity
Recent discoveries have supported the u of small fish models for human health risks from environment and zebrafish has been adopted as one of the model organism animals for the study of environmental toxicity by the NIEHS of USA(for review,e Ref.28).Dibenzo-p-dioxins,for example,are persistent,toxic and widely distributed environmental chemi-cals.Fish embryos and zebrafish are among the most-nsitive organisms,showing vere defects after exposure to TCDD(2,3,7,8-tetrachlorodibenzo-p-dioxin).TCDD significantly incread cell death in the dorsal mid
brain and decread blood flow in the mencephalic vein,one of the earliest-occurring effects of TCDD in zebrafish embryos.(29,30) Since the effects could be mimicked by exposure to beta-naphthoflavone,an agonist of the arylhydrocarbon(ArH) receptor,and inhibited by alpha-naphthoflavone,the authors hypothesized that the TCDD-induced apoptosis is caud through ArH-mediated mechanisms.Concomitant exposure to TCDD and either an inhibitor of cytochrome P450(SKF525A or miconazole)or an antioxidant(N-acetylcyteine or ascorbic acid)also inhibited the TCDD-induced apoptosis and the decrea in blood flow in the mencephalic vein.The results suggest that TCDD-induced oxidative stress was associated with CYP1A induction and likely the reason for the obrved defects.A general caspa inhibitor,Z-VAD-FMK,specifically and strongly suppresd the induction of TCDD-induced apoptosis,if supplied to the medium bathing
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