Model Systems and Organisms Model Systems and Organisms in Toxicologyin ToxicologyThomas et al. EHP (2002)Parallelogram ApproachParallelogram Approachto Characterize Toxicityto Characterize ToxicityWhy Use Models?Why Use Models?Very limited number of studies can be done on humansAllows for controlled experimentsEnvironmental variables can be controlledDosage/route of exposures can be controlled/variedExperiments can be replicatedPhysiology/anatomy can be matched to humansCommonly Used ModelsCommonly Used ModelsUnicellular organisms:Unicellular organisms:• e.g., bacteria, yeast• basic cellular questions• cannot use for multi-cellular interactionsMulticellularMulticellularnonnon--vertebrates:vertebrates:• e.g., flies (Drosophila), worms (C. elegans)• pathway analysis• only rudimentary physiology, very different from humansyeastbacteriaplantsfliesGenotoxicity Testing in Lower OrganismsSubstitution mutationsFrameshiftmutationsThe purpose of the bacterial reverse mutation assay (Ames test) is to evaluate a chemical's genotoxicity by measuring its ability to induce reverse mutations at selected loci in several bacterial strains. This assay measures genetic damage in DNA by using five or more tester strains of bacteria. The Salmonella typhimurium and Escherichia coli strains used in the assay each have a unique mutation that has turned off histidinebiosynthesis in Salmonella or tryptophanbiosynthesis in E. coli.Because of these original mutations, the bacteria require exogenous histidine or tryptophan to survive and will starve to death if grown without these essential nutrients. The key to the assay is the bacteria can undergo a reverse mutation turning the essential gene back on permitting the cell to grow in the absence of either histidine or tryptophan. Each bacterial strain was created by a specific type of mutation - either a base-pair substitution or frame-shift mutation. Because a reverse, compensating mutation usually must occur by the same mechanism, mechanistic toxicology information is also available from Ames assay results based on the pattern of which strain(s) reverted. Image: www.bioreliance.com/AMES_assay.htmlDescription: www.bioreliance.com/AMES_assay.htmlImage: www.mun.ca/biology/scarr/Image: www.mun.ca/biology/scarr/Non-mammalian vertebrates:• e.g., fish (Zebrafish), frogs (Xenopus)• pathway and developmental analysis• differ significantly in physiology from humansNon-primate mammals:• e.g., mouse (Mus), rat (Rattus)• more physiological• physiology similar, but not identical to humans• can be engineered to match humansCommonly Used ModelsCommonly Used ModelsLagadic and Caquet (1998)Non-human primates:• e.g., baboons• primate-specific analysis• very similar in physiology to humans• very expensive and raises ethical issuesIn vitro models:• e.g., cells (primary/cell lines), organ slices, organelles, proteins, etc.• basic mechanistic (cellular?) questions• some degree of cell-cell interactions• less expensive than animal models• limited number of ethical concerns• THE FUTURE OF TOXICITY TESTINGCommonly Used ModelsCommonly Used ModelsUse of Laboratory Animals in ResearchUse of Laboratory Animals in Research• Veterinarians are toxicologists' best friends!• Understanding of laboratory animals' biology, physiology is far from complete• There is a wide variety of species from which the toxicologist may choose (background data, biological characteristics, cost, sensitivity, etc.)• Quality of published information• Public interest to animal research (PETA)• Ethical and moral responsibility for the lives of animals used in research• Responsible use of animals: 3 "R"s (Refinement, Reduction, Replacement)Use of Laboratory Animals in ResearchUse of Laboratory Animals in Research• All research using animals must adhere to scientific, institutional, and governmental principles, policies, laws, regulations and guidelines• Ethical and moral responsibility of each researcher for the lives of animals• Researchers are responsible for:• Quality of animal care• Appropriateness of animal use• Minimization or relief of pain and distressRegulations, Laws, Policies and GuidelinesRegulations, Laws, Policies and Guidelines• Guidelines and Recommendations:Developed by independent groups (e.g., Association for Assessment and Accreditation of Laboratory Animal Care [AAALAC]), are not regulated by law, but can (and are!) be included in an overall policy that governs institutional activities, or eligibility to receive funding• Laws and Regulations:require mandatory compliance, failure to comply is enforced by legal actions (fines, revocation of rights to use animals, imprisonment)USDA administers laws and regulations on use of animals:registration, inspections, control, preventionUSDA Æ Animal, Plant and Health Inspection Service (APHIS) ÆRegulatory Enforcement and Animal Care (REAC)Regulations, Laws, Policies and GuidelinesRegulations, Laws, Policies and Guidelines• The Animal Welfare Act (1966)• The Guide for the Care and Use of Laboratory Animalsis a guideline, not a law, was developed by NAS, used as a reference for voluntary assurance and accrediting bodies such as AAALAC and NIH's Office for Laboratory Animal Welfare (OLAW)• Institutional Animal Care and Use Committeeis responsible for evaluation and oversight of the institution's animal care and use program and all related issues set forth in The Guide:Inspects animal facilities and laboratories where animals are used;Carries out programmatic reviews of individual research programs;Recommends actions to be taken by investigators and/or officials;Reviews and approves protocols for animal use in research.Alternatives to Animal Use in ResearchAlternatives to Animal Use in Research• In Vitro models• Computer-simulated models• Computer structure-activity analysisA suitable replacement for animals should:A suitable replacement for animals should:• Reliably predict biological phenomena• Be at least as good (or better!) and a consistent model for risk assessment in humans as well as in animals• Be extensively validated, tested and accepted by regulatory agencies as suitable substitutesHuman Relevance of Human Relevance of In VitroIn VitroScreensScreensPatientVolunteerAnimal modelTissue/CellsSubcellular FxGenesRELEVANCEMECHANISMAdvantages of Advantages of In Vitro In Vitro Model SystemsModel Systems• Relatively inexpensive, reproducible, and efficient
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