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Experimental Toxicology

Experimental Toxicology. Prof. Dr. Şahan SAYGI NEU Faculty of Pharmacy Department of Toxicology. CONTENTS. Introduction Models for toxicity testing and research The Four R’s In experimental toxicology Animal models in toxicology Current animal studies

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Experimental Toxicology

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  1. Experimental Toxicology Prof. Dr. Şahan SAYGI NEU Faculty of Pharmacy Department of Toxicology

  2. CONTENTS • Introduction • Modelsfortoxicitytestingandresearch • TheFourR’sInexperimentaltoxicology • Animalmodels in toxicology • Currentanimalstudies • Origins of predictiveanimaltesting • Selecting an animal model • Husbandryandcare • Choosingspeciesandstrains • Dosing • Animalphysiology

  3. Introduction • Toxicology is thescienceconcernedwithidentifyingandunderstandingthemechanisms of agentsadverselyaffectingthehealth of humans, otheranimals, andlivingportions of theenvironment. • Toxicology is concernedwiththoseman-madechemicalagentsadverselyaffectingthehealth of humans.

  4. Thecurrent test methodsdesignedandusedtoevaluatethepotential of manmadematerialstocauseharmtothepeople. • On theonehand, oursociety is not onlycriticallydependent on technologicadvancestoimproveormaintainstandards of living, but it is alsointolerant of risks, realorpotential, to life andhealththatareseeminglyavoidable.

  5. On theotherhand, thetraditionaltests (withboththeirmisuseandmisunderstanding of theiruse) haveserved as therallyingpointforthoseindividualsconcernedaboutthehumane, ethical, andproperuse of animals. • Thisconcern has causedalltestingusinganimalstocomeunderquestion on bothethicalandscientificgrounds, and it has provided a continuousstimulusforthedevelopment of alternativesandinnovations.

  6. Since 1980, tremendousprogress has beenmade in ourunderstanding of biologydowntothemolecularlevel. • Thisprogress has translatedintomanymodificationsandimprovements in in vivotestingproceduresthatnowgive us teststhat: • Aremorereliable, reproducible, andpredictive of potentialhazards in humans, • Usefeweranimals • Considerablymorehumanethanareearlier test forms.

  7. Varioustermsareusedtodescribethedifferentkinds of testingandresearchperformedbythe model systemsused. • Invivois usedtodenotetheuse of intacthigherorganisms (vertebrates). • in vitro is usedtodescribethosetestsusingotherthanintactvertebrates as model systems. • Thesetestsincludeeverythingfromlowerorganisms (planariaandbacteria) toculturedcellsandcomputermodels.

  8. Inbetweenclearlyin vivoandin vitromodelsarethe “alternatives.” • Thisterm has a differentmeaningtodifferentpeople. • Initsbroadest sense, it incorporateseverythingthatreduceshigheranimalusageandsuffering in theexistingtraditional test designs.

  9. Thisdefinitionincludesuse of thefollowingrange of situations: • A reducedvolume of test material in a rabbiteyeirritation test • A limited test designtocharacterizelethality in therat • Earthwormsinstead of ratsormiceforlethalitytesting • Fishinstead of ratsormiceforcarcinogenicitybioassays • Computerizedstructureactivitymodelsforpredictingtoxicity • True in vitromodels

  10. Models for toxicity testing and research • Invivo (intacthigherorganism) • Loverorganisms (earthworms, fish) • IsolatedOrgans • Culturedcells • Chemical/biochemicalsystems • Computersimulations

  11. In vivo (intact higher organism) • Advantage; Full range of organismicresponse. • Disadvantage;Costs, ethical/ animalwelfareconcerns, species-to-speciesvariability.

  12. Lover organisms (earthworms, fish) • Advantage:Range of integratedorganismicresponses. • Disadvantage:Frequentlylackresponses of higherorganism, animalwelfareconcerns.

  13. Isolated organs • Advantages: Intactisolatedtissueandvascularsystem, controlledenvironmentalandexposureconditions. • Diadavantages: Donororganismsstillrequired, time consumingandexpensive, nointactorganismsresponses, limitedlenght of viability.

  14. Cultured cells • Advantages: No intactanimalsdirectlyinvolved, abilitytocarefullymanipulatesystem, Lowcosts, widerange of variables can be studied. • Disadvantages: Instability of system, limitedenzymaticcapabilitiesandviability of system, noorlimitedintegratedmulticellororganismicresponses.

  15. Chemical/biochemical systems • Advantages: No donororganismproblems, Lowcosts, long-termstability of preparation, widerange of variables can be studied, specificity of response. • Disadvantages: No de factocorrelationto in vivosystem, limitedtoinvestigation of singledefinedmechanism.

  16. Computer simulations • Advantages: No animalwelfareconcerns, speedandlowper-evaluationcost. • Disadvantages: Problematicpredictivevaluebeyondnarrowrange of structures, Expensivetoestablish.

  17. The Four R’s In Experimental Toxicology • Replacement • Reduction • Refinement • Responsibility

  18. Thefirstandmostsignificantfactorsbehindtheinterest in so-calledin vitrosystemshaveclearlybeenpoliticalcampaignby a widespectrum of individualsconcernedwiththewelfareandhumanetreatment of laboratoryanimals. • Thehistoricalbeginnings of thiscampaignwere in 1959.

  19. Replacement • Using methodsthat do not useintact animals in place of thosethat do. • Forexample, veterinarystudentsmayuse a canine cardiopulmonary-resuscitationsimulator, Resusci-Dog, instead of livingdogs. • Cell culturesmayreplacemiceandratsthatare fed newproductstodiscoversubstancespoisonoustohumans. • Inaddition, usingtheprecedingdefinition of animal, an invertebrate (e.g., a horseshoecrab) couldreplace a vertebrate (e.g., a rabbit) in a testingprotocol.

  20. Reduction • Theuse of feweranimals. • Forinstance, changingpracticesallowtoxicologiststoestimatethelethaldose of a chemicalwith as few as onetenththenumber of animalsused in traditionaltests. • Reduction can alsorefertotheminimization of anyunintentionallyduplicativeexperiments, perhapsthroughimprovements in informationresources.

  21. Refinement • Themodification of existingproceduressothatanimalsaresubjectedtolesspainanddistress. • Refinementsmayinclude ; • administration of anestheticstoanimalsundergoingotherwisepainfulprocedures, • administration of tranquilizersfordistress, • humanedestructionbeforerecoveryfromsurgicalanesthesia, • carefulscrutiny of behavioralindices of painordistress, followedbycessation of theprocedureortheuse of appropriateanalgesics.

  22. Responsibility • Totoxicologists, this is thecardinal R. • Theymay be personallycommittedtominimizinganimaluseandsufferingandtodoingthebestpossiblescience of whichtheyarecapable, but at theend of it all, toxicologistsmuststandbytheirresponsibilityto be conservative in ensuringthesafety of thepeopleusingorexposedtothedrugsandchemicalsproducedandused in oursociety.

  23. ANIMAL MODELS IN TOXICOLOGY • Theuse of animals in experimentalmedicine, pharmacology, pharmaceuticaldevelopment, safetyassessment, andtoxicologicalevaluation has become a well-establishedandessentialpractice. • Animalexperimentsalsohaveservedrathersuccessfully as identifiers of potentialhazardstoandtoxicity in humansforsyntheticchemicalswithmanyintendeduses.

  24. CURRENT ANIMAL STUDIES • Thecurrentregulatoryrequireduse of animalmodels in acutetestingbeganbyusingthem as a form of instrumenttodetectundesiredcontaminants. • Forexample, minersusedcanariestodetectthe presence of carbonmonoxide, a case in which an animal model is moresensitivethanhumans. • In 1907, FDA startedtoprotectthepublicbytheuse of a voluntarytesting program fornewcoal tar colors in foods. Thiswasreplacedbyamandatory program of testing in 1938, andsuchregulatoryrequiredanimaltestingprogramshavecontinuedtoexpanduntilrecently.

  25. TheSociety of Toxicology (SOT) andtheAmericanCollege of Toxicology (ACT) havebothestablishedAnimals in ResearchCommittees, andthesehavepublishedguidelinesfortheuse of animals in researchandtesting. • In general, thepurpose of thesecommittees is tofosterthinking on thefourRsof animal-basedresearch: reduction, refinement, (researchinto) replacements, andresponsibleuse.

  26. Themediafrequentlycarryreportsthatstatethatmostanimaltestingandresearch is not predictive of whatwillhappen in people, andthereforesuchtesting is unwarranted. • Manyanimalrightsgroupsalsopresentthisargument at everyopportunity, andreinforce it withexamplesthatentailseeminglygreatsuffering in animals but addnothingtothehealth, safety, andwelfare of society.

  27. Ourprimaryresponsibility (thefourth R) is toprovidetheinformationtoprotectpeopleandtheenvironment, andwithoutanimalmodelswecannotdischargethisresponsibility. • The problem is thattoxicology is a negativescience. • Thethingswefindanddiscoverareusuallyadverse. • Iftheappliedend of ourscienceworkscorrectly, theresultsarethingsthat do not happen, andthereforeare not seen.

  28. Forexample, ifwecorrectlyidentifytoxicagents (usinganimalsandotherpredictive model systems) in advance of a productoragentbeingintroducedintothemarketplaceorenvironment, generally it will not be introduced (or it will be removed) andsocietywill not seedeath, rashes, renalandhepaticdiseases, cancer, orbirthdefects,.

  29. ORIGINS OF PREDICTIVE ANIMAL TESTING • The “LashLure” Case: • Early in the 1930s, an untestedeyelashdyecontaining p-phenylenediamine (LashLure) wasbroughtontothe market in the United States. • Thisproductrapidlydemonstratedthat it couldsensitizetheexternalocularstructures, leadingtocornealulcerationwithloss of visionand at leastonefatality.

  30. A womanknown as "Mrs. Brown", in 1933, tryingtobeautify her appearancebeforegoingto a socialparty in Dayton , Ohio, wasencouraged in a beautyshoptotry an eyelashdyetoenhance her eyes. • LashLurewasthe name of theproduct. • At thenextmorning, shecouldn'topen her eyes, theywerecompletelyinfected, withulcersandscars, and in threemonthsshebecamepermanentblind.

  31. Advertisements of LashLureEyeLashandBrowDyeweresaying, in 1933, thattheir "newandimprovedmascarawillgiveyou a radiatingpersonality, with a beforeand an after"... • Thislastpartwastrue: the "before" wastheregularappearance, andthe "after" was a horror film, a cosmeticdisaster, withmeltedocularglobes, theflesharoundthemwithmultiplescars, blindedpeoplewithinfectedulcers, and a womandied in thehospitalwithsepticemia, bloodpoisoning.

  32. TheElixir of Sulfanilamide Case: • In 1937, an elixir of sulfanilamidedissolved in ethyleneglycolwasintroducedintothemarketplace. • Onehundredand seven peopledied as a result of ethyleneglycoltoxicity. • ThepublicresponsetothesetwotragedieshelpedpromptCongresstopassthe Federal Food, Drug, andCosmeticAct of 1938. • Itwasthislawthatmandatedthepremarkettesting of drugsforsafety in experimentalanimals.

  33. Thalidomide: • Theuse of thalidomide, a sedative-hypnoticagent, ledtosome 10,000 deformedchildrenbeingborn in Europe. • This in turnleddirectlytothe 1962 revision of theFood, DrugandCosmeticAct, requiringmorestringenttesting. • Currenttestingprocedureswouldhaveidentifiedthehazardandpreventedthistragedy. • Infact, it has not occurred in Europe orthe United Statesexceptwhentheresults of animaltestshavebeenignored.

  34. Forexample, birthdefectshaveoccurredwithisotretinoin (Accutane) wheredevelopmentaltoxicity had beenclearlyestablished in animalsandpresented on labeling, but thedrug has continuedto be usedbypotentiallypregnantwomen. • Isotretinoin is usedfor severe acnetreatment. • Thisdrugshould not be usedbypregnantpatients. • PatientswhohavealreadyusedIsotrotinoin, discontinuethedrugbefore 1 month of earlierandthewholepregnancyperiod.

  35. SELECTING AN ANIMAL MODEL • Choosing the appropriate animal model for a given problem is sometimes guesswork and too often a matter of convenience.

  36. Forexample, theratis probably a poor model forstudyingthechronictoxicity of anynewnonsteroidal anti-inflammatorydrug (NSAID) becausetheacutegastrointestinal (GI) toxicitywillprobably mask anyothertoxiceffects. • Theguineapigis lesssensitivetomostNSAIDsthantherat, andwouldtherefore be a moreappropriatespeciesforinvestigatingthechronic (nongastrointestinal) toxicity of an NSAID.

  37. HUSBANDRY AND CARE • Inappropriatehandlingcouldresult in unhealthyanimalsand an experimentyieldingvariableandirreproducibleresults. • Allanimalshave optimal temperature, humidity, lightcycle, lightintensity, cage size andbedding, anddietaryrequirements.

  38. Rabbits, forexample, have a different optimal temperaturerangethanrats. • Ratsandferretshavecompletelydifferentdietaryrequirements. • Albino rodentshaveverysensitiveeyes, andlights of toohighpower can causeoculardamage, especially in thoseanimals on the top row of a cagerack. FERRET

  39. Caging

  40. Cagingdeservesspecialmentionfortworeasons. First, not allanimals can be grouphoused. • Hamsters, forexample, arenotoriouslyantisocial. Evenbreedingpairscannot be left in thesamesmallcagetogetherforprolongedperiods. • Guineapigs, on theotherhand, flourishwhengrouphoused. Obviouslythesefactorsneedto be consideredwhendesigning an experiment.

  41. Second, cage size is importantbecausetheanimalrightsmovement has made it important. • Manycagingsystemscurrently in usewouldnolonger be permittedandtheirreplacementwould be veryexpensive. • This is just an example of how theanimalrightsmovement, andtheresultantanimalcarelaws, couldaffecttheconduct of pharmacologistsandtoxicologists.

  42. CHOOSING SPECIES AND STRAINS • Not only is it importanttopickthecorrectspeciesfor an experiment, but sometimesthecorrectstrain as well. • Insomecases, an inbredstrainmightprovidequalitativeandspecificcharacteristicsthatmake it a gooddisease model, such as thespontaneouslyhypertensiverat.

  43. Thereareothermorequantitativestrain-relateddifferencessuch as size, color, temperament, and background disease. • Forexample, theFischer 344 rat is smallerthantheSprague-Dawleyrat. • Thesedifferencesmightmake a particularstrainmoreappropriateforoneexperimentthanothers.

  44. TheFischer 344 rat has a high rate of spontaneousLeydigcelltumors as comparedtotheSprague-Dawleyrat, whichwouldmakethelatterlessappropriatefordeterminingif a chemical is a testicularcarcinogen. • Ratsandmiceprovidethegreatestarray of strainsfromwhichtochoose, includingoutbredandsomeinbred.

  45. DOSING • Dosing is theact of introducing a drugorchemicalinto a livingorganism. • Itrequiresactiveinteractionbetweenmanandanimal. • Thereare, however, passivedosingtechniquesthatarealsousedfrequently in whichthechemical is placed in theanimal’sair, water, orfeed, andtheanimaldosesitselfbybreathing, drinking, oreating.

  46. Administering an antibioticintravenously is activedosing; giving it in thefeed is passivedosing. • The main routesusedforactivedosingare oral, intravenous, intraperitoneal, dermal, andsubcutaneous. • Thedoseis the total amount of test articlegiven, such as 1,000 mg. • Thedosageis a rate termand is thedosedividedbytheweight of the test animal; forexample, 1,000 mg/10 kg (for a dog) = 100 mg/kg.

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