Scientific Frontiers in Developmental Toxicology and Risk Assessment (2000) / Chapter Skim
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7 Using Model Animals to Assess and Understand Developmental Toxicity
7 Using Model Animals to Assess and Understand Developmental Toxicity
Pages 151-195
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From page 151... ...
MODEL ORGANISMS AND THE GENETIC APPROACH Single-Cell Organisms Model organisms have been important throughout the study of modern biology. In the 1940s and 1950s, biochemical analysis of bacteria was important in working out the enzymatic pathways of metabolism.
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From page 152... ...
The most commonly studied model animals are relatively inexpensive to maintain and are well suited for experimental manipulation. Most important, as outlined in Chapter 6, recent research has shown that there is a remarkable degree of similarity in the developmental mechanisms of all animals.
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From page 153... ...
The four model animals chosen primarily on the basis of their convenience for genetic analysis are C elegans, Drosophila, zebrafish, and mice.
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From page 154... ...
These systems are described in more detail below, following a brief review of methods in genetic analysis. Rationale and Strategy of the Genetic Approach Genetic analysis has a powerful advantage in that it can "dissect" functionally and define the important components of any biological process without knowing anything about the process in advance simply by isolating mutations that affect it, using those mutations to define the genes that control the process, and then cloning and characterizing those genes and their gene products, thereby revealing molecular mechanisms.
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From page 155... ...
5. Identify additional modifier genes by using suppressor and enhancer screens in a sensitized genetic background for secondary mutations that make the defective phenotype of an existing mutant less or more severe.
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From page 156... ...
Reverse Genetics With the increasing availability of genomic sequence information, the following somewhat different approach is becoming more useful for studying biological processes, especially in organisms such as mammals, for which the forward genetic approach is difficult. It is called reverse genetics, because it starts with a cloned gene of potential interest.
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From page 157... ...
Again, emerging technologies, such as microchips carrying ordered arrays of cDNAs to allow rapid analysis of how a mutation affects mRNA populations, will accelerate and enhance the above approaches. Extrapolation to Humans For many genes identified by forward or reverse genetics in model animals such as the mouse, and particularly for genes relevant to human disease states, the next step is to isolate and characterize the corresponding (orthologous)
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From page 158... ...
Relevant characteristics of each are described briefly below, along with some of their experimental advantages and disadvantages (see also Tables 7-1 and 7-3~. The potential utility of each animal for identifying and investigating mechanisms of developmental toxicants is discussed later in this chapter.
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From page 159... ...
ablation of specific cells has provided information on inductive cell interactions during embryogenesis and larval growth. This knowledge has been extremely useful in analyzing the genetic control of cell-fate determination and the roles of cell signaling pathways by using genetic approaches, as described further below.
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From page 160... ...
Reverse genetics using targeted gene disruption is therefore difficult but can be accomplished by random transposon insertion (Plasterk 1995) or deletion mutagenesis followed by appropriate screens, or it can be accomplished by RNAmediated gene interference (RNAi)
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From page 161... ...
Signaling Pathways in Development Most of the progress in understanding C elegans development has come from application of forward genetics as described above, combined with laser ablation experiments to identify required cell interactions.
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(1998~. cry of the giant polytene salivary gland chromosomes in the 1930s provided a cytological basis for those genetic theorems and thus made Drosophila a key organism for genetic analysis.
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This conservation of genetic structure and function has become the cornerstone of modern developmental biology, forming the basis for the usefulness of model organisms in understanding human developmental mechanisms.
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From page 164... ...
Since this initial screen, screens using sensitized genetic backgrounds have become commonplace in developmental genetics of flies. For example, the initial set of downstream functions of the decapentaplegic pathway (a TGF~ signaling pathway)
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From page 165... ...
Many of the components of the pathway were first discovered in Drosophila by doing selections in mutants in which the pathway was operating close to threshold due to a component of reduced activity. These "sensitized strains" revealed secondary mutations easily, and might be useful for detecting toxicant effects on development.
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From page 166... ...
The Zebrafish History, Biology, and Genetics The zebrafish is a relative newcomer to the list of model animals. It has become important as the first vertebrate to be subjected to large-scale genetic screens, which were shown to be feasible by C
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From page 167... ...
Those mutations, therefore, provide an entrance point to specific pathways of organogenesis. In some cases, the phenotypes resemble congenital disorders, such as aortic coarctation (as noted in Chapter 2, cardiac defects are the most common of live-born human developmental defects)
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From page 168... ...
Because there is extensive synteny (conservation of chromosomal gene order over short distances) even between zebrafish and mammals, the dense EST maps of mouse and human should suggest candidate genes once the map position of an EST is established.
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From page 169... ...
Its use has been broadened as more sophisticated techniques for altering the genome have been developed, and it can now be used to include any animal whose genome has been altered by addition of genetic material or by alteration of existing genes by gene targeting. Transgenic techniques, which were first devised in the 1975-1985 period, have been applied to a variety of experimental animals and agricultural animals, although by far the most common mammalian subject of gene manipulation remains the laboratory mouse.
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From page 170... ...
or the gene itself. Then, a series of transgenic animals is produced with those constructs and assayed for the expression of the reporter gene or gene product.
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171 o .~g .e ca A ~ o ._4 V ¢ ~ z o .~ EM ~ O C)
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Embryonic Stem Cell; Mediated Gene Targeting. A major limitation of the DNA microinjection method for making transgenic animals is that it does not allow the targeted alteration of endogenous genes.
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The targeting construct is similar to that described above for making point mutations. The difference, however, is that a third lox-P site is introduced into a neutral position in the endogenous gene, in addition to the lox-P-flanked selectable marker.
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From page 175... ...
If the transgene happens to integrate in a position that causes the disruption of an endogenous gene, the experiment might be more informative about the endogenous gene than the transgene. With gene targeting, a possible complication can arise if the selectable marker, which is essentially a foreign transgene, remains in the genome.
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In keeping with the third charge to the committee to evaluate how this information might be used to improve qualitative and quantitative risk assessment, this section deals primarily with possible new model organism approaches to toxicant detection and to the analysis of the mechanism of action of toxicants on developmental processes. The committee will draw upon these new approaches in Chapters 8 and 9 in proposing a multilevel, multidisciplinary strategy to improve developmental toxicity assessment.
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From page 177... ...
Using the simple and relatively inexpensive animal model systems amenable to genetics, scientists should be able to answer these questions. If the answers are yes, as is the committee's hypothesis, it should be possible to design evaluation approaches for potential developmental toxicants with the use of animals, having sensitized genetic backgrounds and reporter-gene outputs, to detect effects on specific signaling pathways, as described in further detail below.
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From page 178... ...
A variety of sensitized models and other approaches to assaying effects of known and potential developmental toxicants on specific pathways should be possible in the test animals considered here. All the model animal systems provide opportunities for developing methods of toxicity assessment and for investigating toxicological mechanisms (these opportunities are discussed in greater detail in Chapter 8~.
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Receptor Tyrosine Kinase (RTK) Pathways.
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The pathway of interacting gene products that controls apoptosis during normal C elegans development is well defined (Metzstein et al.
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Genetic analysis has identified components of the pathway that are required for dauer formation under starvation conditions, as well as components required for preventing dauer formation when food is available (Riddle and Albert 1997~. Defective function of either class of components is easily assayed in the laboratory.
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As an approach toward understanding the role of such gene products, their normal functions can be conveniently investigated in C elegans by reverse genetics using the RNAi technique described above.
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Earlier in this chapter, the committee described an example of a screen that uses a sensitized genetic background to identify genes in the receptor tyrosine kinase pathway involved in Drosophilia eye development (Simon et al.
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The eye of a RAS vall2 mutant in which the receptor tyrosine kinase (the sevenless pathway) is overactive.
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From page 185... ...
Indeed, cell lines are available, or could be prepared, with one or the other of the 17 intercellular signaling pathways functioning in culture, and these would be valuable for assessments of toxicant effects (e.g., effects on activin's action as an erythroid differentiation factor in human erythroleukemic cell lines)
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From page 186... ...
It should also be extremely useful for detecting deleterious effect of toxicants on specific behaviors, and as described above, the tests could be done on sensitized strains. Zebrafish The zebrafish is an appropriate model organism in which to test potentially toxic chemicals for several reasons.
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Mouse Transgenic Animals and Developmental Toxicology Mice and rats have long been the mammals of choice for toxicological tests. Their advantage over the previously discussed model organisms is their similarity, as mammals, to humans.
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From page 188... ...
Much more of this analysis can and should be done, both to understand the role of these enzymes in potentiation and detoxification and to define human and mouse differences for better-informed extrapolations of animal-test data. Overexpression Transgenics for the Study of DMEs Another approach to the study of developmental toxicity using transgenic animals is to overexpress, or ectopically express, a gene of interest in the embryo and fetus.
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From page 189... ...
Such abiomarkertransgene could be used for in vivo developmental toxicity studies, or cells from appropriate tissues could be used for in vitro testing. Other stress and checkpoint pathways could be connected to reporter genes as well, as could components of apoptosis.
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From page 190... ...
The importance of modifier genes in the genetic background of each transgenic mouse line cannot be underestimated. It would also be useful to calibrate the tests with animals treated with doses of toxicants just below the level giving detectable structural defects in CNS development.
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From page 191... ...
; short life cycle, so test compounds might persist long enough to complicate the study of sensitive developmental periods and of primary versus secondary effects Dunng pupal development organism is inaccessible to toxicar~ts; short life cycle, so test compounds might persist long enough to complicate the study of sensitive developmental periods and of primary versus secondary effects Relatively expensive, long life cycle, genetics and genom~cs research in progress Relatively expensive, long life cycle, embryonic development in utero and not as easily accessible for manipulation as other model animals Comparative Utility of Model Organisms In summary, each of the four genetically tractable model organisms C elegans, Drosophila, zebrafish, and mouse offers promise for development of tests to identify potential developmental toxicants, and each has advantages and disadvantages, as summarized in Table 7-3.
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From page 192... ...
. The cDNA encoding the allele being studied must first be cloned into an appropriate plasmid vector that includes regulatory sequences that drive and terminate transcription and a selectable marker gene.
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From page 193... ...
by microinjection or by chemical-DNA aggregation methods including calcium phosphate precipitation and liposome-mediated transfection. By using such methods, followed by antibiotic treatment to isolate cells that house the plasmid containing the selectable marker gene, cells can be transfected either transiently or stably.
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From page 194... ...
This allows for the selection of cell types with the proper intracellular accessory factors or proteins for enzyme activities that most closely resemble their in vivo counterparts. Finally, expression of gene products can be controlled by an increasing number of eukaryotic promoters.
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From page 195... ...
The committee begins in this chapter, and continues in the next, the third charge to evaluate how this information can be used to improve qualitative and quantitative risk assessment. In this chapter, the committee summarizes some of the techniques for modifying model organisms, including the mouse, for effective use in assays evaluating agents for potential developmental toxicity and for elucidating mechanisms of toxicity.
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