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Alternatives-to-animal-testing-A-review_2015_Saudi-Pharmaceutical-Journal动物福利-动物实验的第二选择

REVIEW

Alternatives to animal testing:A

review Sonali K.Doke,Shashikant C.Dhawale*

School of Pharmacy,SRTM University,Nanded431606,MS,India

Received13August2013;accepted10November2013

Available online18November2013

KEYWORDS Alternative organism; Model organism;

3Rs;

Laboratory animal; Animal ethics Abstract The number of animals used in research has increased with the advancement of research and development in medical technology.Every year,millions of experimental animals are used all over the world.The pain,distress and death experienced by the animals during scienti?c experi-ments have been a debating issue for a long time.Besides the major concern of ethics,there are few more disadvantages of animal experimentation like requirement of skilled manpower,time con-suming protocols and high cost.Various alternatives to animal testing were proposed to overcome the drawbacks associated with animal experiments and avoid the unethical procedures.A strategy of3Rs(i.e.reduction,re?nement and replacement)is being applied for laboratory use of animals. Different methods and alternative organisms are applied to implement this strategy.These methods provide an alternative means for the drug and chemical testing,up to some levels.A brief account of these alternatives and advantages associated is discussed in this review with examples.An integrated application of these approaches would give an insight into minimum use of animals in scienti?c experiments.

Contents

1.Introduction (224)

2.Three Rs:reduction,re?nement and replacement (224)

2.1.Reduction (225)

2.2.Re?nement (225)

2.3.Replacement (225)

*Corresponding author.Tel.:+919970700030;fax:+912462229245.

E-mail addresses:sonalidoke.raut@https://www.wendangku.net/doc/c018515967.html,(S.K.Doke),

shashiprathmesh@https://www.wendangku.net/doc/c018515967.html,(S.C.Dhawale).

https://www.wendangku.net/doc/c018515967.html,/10.1016/j.jsps.2013.11.002

3.Alternative methods (225)

https://www.wendangku.net/doc/c018515967.html,puter models (225)

3.2.Cells and tissue cultures (226)

3.3.Alternative organisms (226)

3.3.1.Lower vertebrates (226)

3.3.2.Invertebrates (226)

3.3.3.Microorganisms (227)

4.Conclusion (227)

Acknowledgements (228)

References (228)

1.Introduction

Use of animals for various purposes like food,transportation, pets,sports,recreation and companionship is as old as the hu-man beings https://www.wendangku.net/doc/c018515967.html,ing animals for the purpose of research is one of the extended uses.Various animals like mice,rats,ham-sters,rabbits,?shes(examples–zebra?sh,trout),birds (mainly chicken),guinea pigs,amphibians(xenopus frogs), primates,dogs,cats etc.are being used in research for a long time(CULABBR,1988).Drug testing and toxicological screenings which are useful in the development of new treat-ments for infectious and non-infectious diseases is the main purpose of such studies.Animals also serve as a tool to under-stand effects of medical procedures and surgical experiments. Moreover,they are used to obtain products like vaccines,anti-biotics etc.which are used in diagnostics as well as treatments (Giacomotto and Segalat,2010;Hendriksen,2009,2007).The number of animals used in research has gone up with the advancement in medical technology.Every year,millions of experimental animals are used all over the world.For example, in UK,3.71million animals were used for research in the year 2011(https://www.wendangku.net/doc/c018515967.html,).The total number of animals used in the USA in the year2009was estimated to be1,131,076,while that in Germany reached up to2.13million in2001(Rusche, 2003).This huge population of experimental animals usually comes from the breeding centers located in various universities and national breeding centers.All of these are known as class-A dealers,while the brokers who acquire the animals from mis-cellaneous sources(like auctions and animal shelters)are iden-ti?ed as class-B dealers.At few instances use of the wild animals such as monkeys and birds is also followed(Baumans, 2005).In clinical testing laboratories,animals are isolated from their groups and used as a tool irrespective of their natural in-stincts.For the experimental procedures,either a whole animal or its organs and tissues are used.For this purpose animals are euthanized(killed)by established methods.Many times,the animals surviving the clinical testing are euthanized at the end of an experiment to avoid the later pain and distress (Rusche,2003).In some cases(for example in LD50analysis) animals die as a result of the experiment.

The pain,distress and death experienced by the animals during scienti?c experiments have been a debating issue for a long time.Argument is that being alive,animals have the rights against pain and distress and hence,their use for exper-imentation is unethical and must be stopped(Rollin,2003). Various acts and laws have been passed to bring the control over unethical use of animals and minimize the pain to animals during experimentation.For example,in1824,the organiza-tion for animal rights was formed by the Royal Society for the Prevention of Cruelty to Animals.In1876,an act for pre-vention of cruelty to animal was formed in the UK(Balls, 1994).It came into existence in India,France and USA in the year1960,1963and1966,respectively.At present,many rules and acts are followed at the international level,to protect the animals against the cruelty and misuse.The organizations like ICH(International Conference on Harmonization of tech-nical requirements for registration of pharmaceuticals for hu-man use),CPCSEA(Committee for Purpose of Control and Supervision on Experiments on Animal),NIH(National Insti-tute of Health),and OECD(Organization for Economic Co-operation and Development)provide the guidelines for animal house keeping,breeding,feeding,transportation,and mainly for their use in scienti?c experiments(Rollin,2003).Besides the major concern of ethics,few more disadvantages of animal experimentation are requirement of skilled/trained manpower and time consuming protocols.Moreover,very high cost in-volved in breeding,housing and lengthy protocols of animal experiments is another drawback(Balls,1994).

2.Three Rs:reduction,re?nement and replacement Alternatives to animal testing were proposed to overcome some of the drawbacks associated with animal experiments and avoid the unethical procedures.A strategy of3Rs is being applied which stands for reduction,re?nement and replace-ment of laboratory use of animals(Ranganatha and Kuppast, 2012).Different methods and alternative organisms are ap-plied to implement this strategy.The concept of replacement of animals was?rst discussed in1957by Charles Hume and William Russell at the Universities Federation for animal wel-fares(UFAW)(Balls,1994).Russell and Burch(1959)sug-gested some ways to make the animal experiments more humanly,which was later called as3Rs.This approach moti-vates the use of minimum number of animals i.e.‘reduction’in the total number of animals used in an experiment.The use of animals must be planned and‘re?ned’carefully in such a way that pain and distress caused during the experiment should be minimized.Moreover,if possible higher animals should be‘re-placed’with alternative methodologies and lower organisms (Ranganatha and Kuppast,2012;Zurlo et al.,1996).Animal replacement is de?ned as,‘any scienti?c method employing non-sentient material which may replace use of conscious liv-ing vertebrates in animal experimentation’.Two types of replacements were distinguished as‘relative’and‘absolute’replacement.In relative replacement the animals are used but not exposed to any distress during experiment.No use of

224S.K.Doke,S.C.Dhawale

animals at any stage of experiment is identi?ed as the absolute replacement strategy(Balls,1994).

2.1.Reduction

With the help of statistical support and careful selection of study design one can produce meaningful scienti?c results of an experiment.For example,in vitro cell culture is a good way to screen the compounds at early https://www.wendangku.net/doc/c018515967.html,e of the human hepatocyte culture gives the information about how a drug would be metabolized and eliminated from the body.Inclusion of such method in study design helps to eliminate unsuitable compounds in preliminary stages only and minimizes the use of animals in further tastings(Kimber et al.,2001).Live ani-mals and embryos are used to study effects of some com-pounds on embryo development.In vitro embryonic stem cell culture test helps to reduce the number of live embryo used and the compounds which are toxic toward developing embryo (Gipson and Sugrue,1994;De Silva et al.,1996).Also,sharing or providing the discovered data(like characteristics of excip-ients for the test drug)avoids the necessity of animal studies.

2.2.Re?nement

Enriching the cage environment by taking care of animals reduces the stress on animals.Scientists should re?ne the ani-mal facility so that pain,discomfort and distress during animal life and scienti?c procedures are reduced.Moreover,under the stress and discomfort there may be imbalance in hormonal levels of animals leading to?uctuations in the results.Hence, experiments need to be repeated which causes an increase in the number of experimental animals.So re?nement is neces-sary not only to improve the life of laboratory animals but also to improve the quality of research(Hendriksen,2009).For example,it was observed that when mice genetically modi?ed to study Huntington’s disease were provided with a complex cage environment with opportunity to nest,hide,gnaw and forage,the disease progressed slowly than the mice in barren cage.Also,such mice were found to mimic the progress of the human disease more closely.Such a re?nement provides a very good model to treat the disease and also minimize stress to the animals(De Silva et al.,1996).

2.3.Replacement

Various alternatives to the use of animals have been suggested, such as in vitro models,cell cultures,computer models,and new imaging/analyzing techniques(Balls,2002).The in vitro models provide the opportunity to study the cellular response in a closed system,where the experimental conditions are maintained.Such models provide preliminary information for outcome of an experiment in vivo.For example,computer models were used to study the working of the heart and to select the potential drug candidates(Gipson and Sugrue, 1994).In many countries,in vitro cell cultures have replaced the skin irritancy test and Draize eye irritancy test and use of animals in those.Another example is,extraction of insulin from the pancreas of pigs and cow,but now it is obtained from the bacterial cultures which are lifeline drugs for diabetic patients.This extracted insulin needs to be checked for its purity,ef?cacy and https://www.wendangku.net/doc/c018515967.html,e of animals was routine for such checking,but now chromatography techniques are used for checking the purity,ef?cacy and calculation of dosages of drugs(Foreman et al.,1996).Overall,replacement substan-tially reduces the use of animals in various processes.

3.Alternative methods

Various methods have been suggested to avoid the animal use in experimentation.These methods provide an alternative means for the drug and chemical testing,up to some levels. Advantages associated with these methods are,time ef?ciency, requires less man power,and cost effectiveness.These methods are described in detail as follows-

https://www.wendangku.net/doc/c018515967.html,puter models

Computers can help to understand the various basic principles of biology.Specialized computer models and software programs help to design new https://www.wendangku.net/doc/c018515967.html,puter generated simulations are used to predict the various possible biological and toxic effects of a chemical or potential drug candidate without animal dissection.Only the most promising molecules obtained from primary screening are used for in vivo experimentation.For example,to know the receptor binding site of a drug,in vivo experimentation is necessary.Software known as Computer Aided Drug Design(CADD)is used to predict the receptor binding site for a potential drug molecule.CADD works to iden-tify probable binding site and hence avoids testing of unwanted chemicals having no biological activity.Also,with the help of such software programs we can tailor make a new drug for the speci?c binding site and then in?nal stage animal testing is done to obtain con?rmatory results(Vedani,1991).Hence,the total number of experimental animals is lowered and the objectives of Russel and Burche’s3Rs are achieved.

Another popular tool is the Structure Activity Relationship (SARs)computer programs.It predicts biological activity of a drug candidate based on the presence of chemical moieties at-tached to the parent compound.Quantitative Structure Activ-ity Relationship(QSAR)is the mathematical description of the relationship between physicochemical properties of a drug molecule and its biological activity(Knight et al.,2006).The activities like carcinogenicity and mutagenicity of a potential drug candidate are well predicted by the computer database. The recent QSAR software shows more appropriate results while predicting the carcinogenicity of any molecule.The advantages of computer models over conventional animal models are the speed and relatively inexpensive procedures (Matthews and Contrera,1998).A very good example is a study by Dewhurst et al.(1994)which assessed the effective-ness of computer models versus the traditional laboratory practices.In this comparative study,two groups of undergrad-uate students performed an experiment with the traditional wet lab approach and computer assisted learning(CAL),respec-tively.CAL is an interactive computer assisted learning (CAL)program without involvement of real experimental tools.At the end of the study both the groups were assessed for the knowledge gain(through test questionnaires,calcula-tions,and interpretation).It was found that the students performing CAL had a better problem solving attitude. Moreover,the cost of new techniques was much less than the traditional laboratory practices(Dewhurst et al.,1994).

Alternatives to animal testing:A review225

3.2.Cells and tissue cultures

Use of in vitro cell and tissue cultures which involves growth of cells outside the body in laboratory environment can be an important alternative for animal experiments.The cells and tis-sues from the liver,kidney,brain,skin etc.are removed from an animal and can be kept outside the body,in suitable growth medium,for few days to several months or even for few years. In vitro culture of animal/human cells includes their isolation from each other and growing as a monolayer over the surface of culture plates/?asks.Cellular components like membrane fragments,cellular enzymes can also be used.Various types of cultures like cell culture,callus culture,tissue culture and or-gan culture are used for various purposes.Bene?ts associated with techniques are,easy to follow,less time consuming and are less expensive.These methodologies are routinely used for preliminary screening of potential drug molecules/chemi-cals to check their toxicity and ef?cacy(Shay and Wright, 2000;Steinhoff et al.,2000).Almost all cosmetics,drugs and chemicals are tested for their toxicity and ef?cacy,using these tests.For example,eye irritancy test.To check the irritancy of chemicals previously Draize test was used,which requires ani-mals(mainly rabbit).It is very painful and every time a new animal is used.Ke Ping Xu and coworkers suggested an alter-native which uses bovine corneal organ culture.The bovine cornea is cultured up to three weeks in laboratory and various analytical methods are used to evaluate the toxicological effect of test chemical irritancy in vitro(Xu et al.,2000).

3.3.Alternative organisms

The ethical issues have posed many restrictions over the exper-imental use of higher model vertebrates like guinea pig,rats, dogs,monkeys etc.Therefore,use of alternative organisms has been proposed.Different model organisms are used to re-place experimental animals(Table1).3.3.1.Lower vertebrates

Lower vertebrates are an attractive option because of the genetic relatedness to the higher vertebrates including mam-mals.Moreover,there are less ethical problems involved in the experimental use of lower vertebrates.

3.3.1.1.Example–Danio rerio.Danio rerio,commonly called as zebra?sh,is a small freshwater?sh with an approximate length of2–4cm.It has a nearly transparent body during early development,which helps easy visual access to the internal anatomy.The optical clarity allows direct observation of developmental stages,identi?cation of phenotypic traits dur-ing mutagenesis,easy screening,assessment of endpoint of tox-icity testing and direct observation of gene expression through light microscopy.Small size,short life cycle and high fecundity favor its laboratory use.

The working space,cost of laboratory solutions,test chem-icals and the manpower involved are reduced by opting D.re-rio as an alternative to animals(Hill et al.,2005).Its embryos and larvae can be developed and used for testing in cell culture plates and Petri dishes.Whole genome sequence availability makes Zebra?sh an attractive option for molecular and genet-ic research.From infancy to the adult stage it is used in a vari-ety of applications,mainly for the detection of various toxicological studies of chemicals and pharmaceuticals.It is also having wide applications in the investigation of cancer, heart diseases,neurological malfunctions,behavioral diseases and to observe the mutations and problems in organ develop-ment due to exposure to test molecules.Modeling of certain human diseases in zebra?sh could be used to ameliorate the disease phenotype and malfunctions in organ development (Peterson et al.,2008).

3.3.2.Invertebrates

Invertebrate organisms are widely used as an alternative for laboratory use of animals.They have been used to study

Table1Selected examples of organisms as alternatives for laboratory use of animals.

Alternative organism Remarks

Prokaryotes

Escherichia coli Model for molecular and genetic studies

Bacillus subtilis Model for cellular di?erentiation

Caulobacter crescentus

Protists

Dictyostelium discoideum Model for molecular and genetic studies

Fungi

Neurospora crassa Model for genetic study,circardian rhythm and metabolic regulation studies Saccharomyces cerevisiae

Schizosaccharomyces pombe Model for molecular and genetic studies

Aspergillus nidulans

Lower vertebrate

Danio rerio/zebra?sh

Invertebrates

Amphimedon queenslandica Studies on evolution,developmental biology and comparative genomics Aplysia sp./sea slug Neurobiology

Caenorhabditis elegans Genetic development studies

Drosophila melanogaster Genetics and neurology research

Hydra/Cnidaria To understand the process of regeneration and morphogenesis

226S.K.Doke,S.C.Dhawale

various diseases like Parkinson’s disease,endocrine and mem-ory dysfunction,muscle dystrophy,wound healing,cell aging, programmed cell death,retrovirus biology,diabetes and toxi-cological testing(Lagadic and Caquet,1998).Invertebrates have an undeveloped organ system and do not have the adap-tive immune system,which poses some limitations for their use in human diseases.However,they hold numerous bene?ts, such as a brief life cycle,small size and simple anatomy,so that a large number of invertebrates can be studied in a single experiment within a short period with less ethical problems. Their cost of housing is less compared to the animals.For example,thousands of?ies could be accommodated in a shel-ter where only few mice can be kept(Wilson-Sanders,2011).

3.3.2.1.Example–Drosophila melanogaster.Drosophila mela-nogaster,also known as fruit?y is one of the most widely stud-ied invertebrates in research(Gilbert,2008).It has a well studied genome which enables study of molecular mechanisms underlying the human diseases.Its complete genome has been sequenced and annotated,which encodes more than14,000 genes on four chromosomes.Only three genes carry the bulk of genome of D.melanogaster.Nearly75%of the genes in-volved in human diseases are believed to have a functional homolog in the?y(Reiter et al.,2001;Wilson-Sanders, 2011).D.melanogaster requires extremely low cost of mainte-nance,propagation and screening as compared to the other mammal based models.It also produces the results very rap-idly due to a short life cycle.Fruit?y possesses four stages in life cycle–the embryo,the larva,the pupa and the adult. Each stage of?y has its own advantage,hence considered as a multiple model organism to study the various concepts(Pan-dey and Nichols,2011).The Embryo is frequently used to study the cell fate determination,neuronal development,axon path?nding,organogenesis,fundamental developments and to examine pattern formation.The larva is used to study the physiological and developmental processes and behaviors like foraging.The adult?y is a very complex organism.The func-tions of various structures like the heart,lungs,gut,kidney and reproductive tract are equivalent as that of mammals(Rothen-?uh and Heberlein,2002).

The response of?ies to many drugs which are acting on CNS is similar to that observed in mammals.The brain of the adult?y is quite extraordinary because more than 100,000neurons form the discreet circuits,which mediate var-ious complex behaviors like circadian rhythms,learning and memory,feeding,sleep,courtship,aggression,grooming and ?ight navigation(Pandey and Nichols,2011;Rothen?uh and Heberlein,2002;Wolf and Rockman,2008).Number of molecular and genetic tools has been made available to study Drosophila.Due to many similarities in development and behavioral activities,fruit?y served as a unique and sensitive model for the study of human genetics and diseases(Becking-ham et al.,2005).It is also used to express the protein products found in human diseases and to compare the resulting patho-logic conditions.Fruit?y serves as an important tool to inves-tigate neurodegenerative diseases like Alzheimers,Parkinson’s, disease and Huntington’s disease(Bonini and Fortini,2003; Iijima and Iijima-Ando,2008;Iijima et al.,2004).It is used in primary small molecule discovery validation as well as in the target discovery processes by taking advantage of the sophisticated genetics available in it.In1994the Nobel Prize for physiology and medicine was awarded to Ed Lewis for his pioneering research de?ning gene structure in?ies,as well as to Eric Weischaus and Christiane Nusslein-Volhard for their studies investigating embryogenesis(Iijima et al.,2004).

3.3.2.2.Example–Caenorhabditis elegans.Caenorhabditis ele-gans is a eukaryotic nematode.This multi cellular organism is approximately1mm in length and has a very short generation https://www.wendangku.net/doc/c018515967.html,plete life cycle of this hermaphrodite is about2–3weeks.Embryogenesis occurs in12h and an adult form is developed in2.5days.It is transparent,genetically amenable and has simple cellular complexity.Hence,was selected as a model organism by Nobel laureate Brenner(Barr,2003; Strange,2007).Life cycle of C.elegans proceeds through var-ious complex developmental stages like embryogenesis,mor-phogenesis and growth to an adult.This is one of the most commonly used model organisms for research https://www.wendangku.net/doc/c018515967.html,r-mation obtained can be applicable to more complex organisms like humans.As a model,C.elegans have been used to study various neurological disorders like Huntington’s disease,Par-kinson’s disease,Alzheimer’s diseases;various immune disor-ders as well as cancer,diabetes.It has served development and testing of the therapeutic agents for treatment of these dis-eases(Artal-Sanz et al.,2006;Faber et al.,1999;Link et al., 2001;Nass et al.,2008;Pujol et al.,2008).

3.3.3.Microorganisms

3.3.3.1.Example–Saccharomyces cerevisiae.Brewing yeast, Saccharomyces cerevisiae is the most popular and important model organism due to its rapid growth,ease of replica plating and mutant isolation,dispersed cells,well de?ned genetic sys-tem and highly versatile DNA transformation system.Yeasts can be grown in solid or liquid culture and isolated as colonies derived from a single cell on solid media.The generation time is very short i.e.about90min,hence it is very easy to grow a large population and analyze it(Mell and Burgess,2002). Whole genome of this unicellular fungus has been sequenced in1996.The nuclear genome contains about16chromosomes with more than13million base pairs.It also contains an extra nuclear genome in the mitochondria.The budding yeast carries its genetic information in the form of6000genes.The number and size of genes are relatively small and the density of genes is very high.Best characterized and studied genome makes S. cerveisiae one of the most ideal eukaryotic microorganisms for the biological studies.Presence of similar cellular architec-ture and rudimentary life cycle like multi cellular eukaryotes is another advantage.The numerous membrane-bound organ-elles like nucleus,peroxisome,mitochondria and the organelles of secretary pathway also mimic the functions of mammalian cells(Mell and Burgess,2002).This brewing yeast is used to understand programmed cell death,cell death regulators in hu-mans and is very useful in cancer research(Madeo et al.,2002). S.cerevisiae helps to understand the fundamental aspects of cellular biology in neurodegenerative diseases like Alzheimer’s, Parkinson’s and Huntington’s diseases by studying the endog-enous or heterologous proteins that lay at the root of these dis-eases(Pereira et al.,2012;Siggers and Lesser,2008).

4.Conclusion

Animal ethics is an issue as important as the human welfare. More efforts need to be undertaken for effective implementation

Alternatives to animal testing:A review227

of3Rs during laboratory use of animals.Various alternatives to animal use have been suggested,which need to be implemented in an effective manner.For this integration of various computer models,bioinformatics tools,in vitro cell cultures,enzymatic screens and model organisms are https://www.wendangku.net/doc/c018515967.html,e of modern ana-lytical techniques,data acquisition and statistical procedures to analyze the results of alternative protocols can provide depend-able outcomes.These integrated approaches would result in minimum involvement of animals in scienti?c procedures. Acknowledgements

Authors are thankful to Prof.S.Mohan Karuppayil,former Director,School of Life Sciences,SRTM University for his kind support.

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