Model Systems and Organisms in Toxicology Thomas et al EHP 2002 Parallelogram Approach to Characterize Toxicity Why Use Models Very limited number of studies can be done on humans Allows for controlled experiments Environmental variables can be controlled Dosage route of exposures can be controlled varied Experiments can be replicated Physiology anatomy can be matched to humans Commonly Used Models Unicellular organisms e g bacteria yeast basic cellular questions cannot use for multi cellular interactions Multicellular non vertebrates e g flies Drosophila worms C elegans pathway analysis only rudimentary physiology very different from humans Commonly Used Models Non mammalian vertebrates e g fish Zebrafish frogs Xenopus pathway and developmental analysis differ significantly in physiology from humans Non primate mammals e g mouse Mus rat Rattus more physiological physiology similar but not identical to humans can be engineered to match humans Commonly Used Models Non human primates e g baboons primate specific analysis very similar in physiology to humans very expensive and raises ethical issues In 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 TESTING Use 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 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 distress Regulations 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 prevention USDA Animal Plant and Health Inspection Service APHIS Regulatory Enforcement and Animal Care REAC Regulations Laws Policies and Guidelines The Animal Welfare Act 1966 The Guide for the Care and Use of Laboratory Animals is 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 Committee is 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 Research In Vitro models Computer simulated models Computer structure activity analysis 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 substitutes Human Relevance of In Vitro Screens Patient RELEVANCE Volunteer Animal model Tissue Cells Subcellular Fx Genes MECHANISM Advantages of In Vitro Model 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 reduce 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 structureactivity relationships Limitations of In Vitro Model 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 Vitro Systems for Studying Hepatotoxicity Isolated perfused liver Liver slices Hepatocytes suspensions cultures Microsomes S9 sub fractions Plasma membrane vesicles Expressed hepatic proteins P450 s transporters receptors Systems for Studying Liver Enzyme Induction In vivo animal studies ex vivo analysis Liver slices Primary cultures of hepatocytes Cell lines HepG2 Huh7 Nuclear receptor assays cell lines stably or transiently expressing appropriate transcription factors and reporter genes Binding assays with specific nuclear receptors In Vitro Procedures for Testing Chemicals as P450 Enzyme Inducers LIVER SLICES In precision cut liver slices the extracellular matrix and cell cell communications are preserved Cells remain viable for several days and are excellent systems for studying phase I and II biotransformation for up to 12 hours but P450 activity declines rapidly after the first 24 48 hours There are limited data on hepatocellular morphology and function i e liver specific gene expression in long term cultures Liver slices are initially refractory to P450 inducers and are not very responsive compared with cultured hepatocytes and the situation in vivo In Vitro Procedures for Testing Chemicals as P450 Enzyme Inducers