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 humansNon-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 ModelsNon-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 ways to investigate toxicity at the cellular and molecular level.• Facilitate the study of mechanisms of toxicity in specific cell types. • Allow for rigorous control and easy manipulation of the experimental conditions.• Significantly reduces the number of animals required for research.• Provide a means for predicting the interaction and toxicity of xenobiotics with human-relevant systems.• Provide rapid and effective means of screening and ranking chemicals.• Are essential for bridging between experimental animals and humans, and for detailed understanding of the bases of species differences.• Provide well-defined systems for studying structure-activity relationships.Advantages of Advantages of In Vitro In Vitro Model SystemsModel SystemsLimitations of Limitations of In Vitro In Vitro Model SystemsModel Systems• Not the “real” thing. • Typically a “static” or “non-native” model system.• Systematic loss of phenotypic properties or functions of original tissue and cells.• Similar liabilities with immortalized cell lines.• In vivo relevance often decreases with the simplicity of the model system.In VitroIn VitroSystems for Studying Systems for Studying HepatotoxicityHepatotoxicity• Isolated perfused liver• Liver slices• Hepatocytes (suspensions, cultures)• Microsomes/S9 sub-fractions• Plasma membrane vesicles• Expressed hepatic proteins - P450’s, transporters, receptorsSystems for Studying