Animal Models in Toxicologic Research: Nonmammalian

Basic Biologic Characteristics and Common Species

Aquatic invertebrates such as hydra and water fleas Daphnia magna have been used for centuries as subjects for basic biological research. Until recently, however, toxicological testing in invertebrates often involved simple exposure studies in which behavioral changes and percent mortality were the predominant endpoints. Today, this is no longer the case, as exemplified by the ever-expanding use of invertebrates such as Drosophila melanogaster fruit flies and Caenorhabditis elegans nematodes for both mechanistic and high-throughput screening studies in the areas of neurotoxicology, environmental toxicology, and genetic toxicology. In fact, representatives from many invertebrate phyla are currently used in pharmaceutical and environmental toxicological research, and examples include Porifera (e.g., marine sponges), Cnidaria (e.g., hydra, corals and anemones), Nematoda ( C. elegans ), Mollusca (e.g., snails, bivalves, and cephalopods such as Octopus vulgaris and squid), Platyhelminthes (e.g., planaria), Arthropoda (e.g., fruit flies, horseshoe crabs, and crustaceans such as daphnia and mysid shrimp), and Echinodermata (e.g., sea urchins) ( ).

The potential for health threats to native and cultivated bee populations and other pollinators to impact commercial agriculture is becoming ever more apparent. Nontoxic threats to these insect populations include infectious agents such as Varroa destructor mites, Nosema apis microsporidian organisms, hive invaders such as Aethina tumida beetles, and losses to starvation, queen failure, and the poorly understood and likely multifactorial condition known as “colony collapse disorder” ( ). Additionally, however, there is concern that exposure of bees to natural and anthropogenic substances such as insecticides (e.g., neonicotinoids, acetylcholinesterase inhibitors), herbicides (e.g., paraquat), fungicides (e.g., chlorothalonil), and other environmental contaminants ( ) [ see Agro/Bulk Chemicals , Vol 2, Chap 12 and Environmental Toxicologic Pathology , Vol 2, Chap 18 ] may have negative health effects, and may be especially hazardous for native pollinators, which, unlike commercially reared honey bees ( Apis mellifera ), cannot be readily replenished. Bees may be exposed to such chemicals intentionally or inadvertently via a variety of mechanisms, including the deliberate use of insecticides to control bee pests, products used to maintain the structural integrity of hives (e.g., tin- and arsenic-based wood preservatives), pesticide overspray, and contamination of pollen and dusts transported to the hive by resident or visiting bees ( ). The list of potential bee toxicants that have been investigated to date is long and continues to grow ( ). Although bee histopathology is still far from routine, recommended techniques for processing tissues from these and other insects have been recently published ( ).

There are a number of intuitive advantages to the use of invertebrates as test subjects, which include the relatively low cost of the animals, housing, equipment, and amounts of test reagents, small space requirements, rapid generation times, exquisite sensitivity to many toxicants (e.g., metals), and comparatively less animal use concern. Additionally, many invertebrate models offer unique anatomical or physiological attributes that can be exploited in toxicological investigations, such as the giant axons of squid, the retinal neurons and well-characterized immune systems of horseshoe crabs, the regenerative capability of planarians, and ability of bivalve mollusks to filter and bioaccumulate contaminants. For example, the inability of efflux transporters belonging to the ATP Binding Cassette superfamily to recognize and export perfluorocarbons has been studied in mussels ( ), and potential links have been investigated between contaminants such as dioxins and unusually high regional prevalences of gonadal tumors in Mya arenaria soft-shell clams ( ).

A primary limitation of invertebrate models is their relative genetic, anatomic, and physiologic distance from humans. For example, some phyla lack complex organs comparable to those of the mammalian immune, cardiovascular, and urinary systems. However, in many cases, this shortcoming is eclipsed by the lower cost and distinctive features of many invertebrate models, and the opportunity they provide for institutions to meet their goals of “reducing, refining, and replacing” mammalian assays.

Background Findings with Examples of Target Tissue Toxicities

Lists of background findings have not yet been compiled for the multitude of currently used and potential invertebrate test subjects. In contrast to traditional toxicologic bioassays, histopathology is less commonly employed in invertebrate toxicity testing, where typical endpoints have traditionally included lethality, behaviors such as stimulus avoidance, and biochemical, electrochemical, or molecular analyses. Morphologic studies that are performed in invertebrate studies may instead rely on external visualization of internal tissues, or, because of the minute nature of some invertebrate organs, ultrastructural evaluation. Histopathology is more likely to be used to investigate outbreaks of infectious disease in economically important invertebrates, such as edible shellfish, and ecologically pivotal animals such as corals, in which secondary bacterial or fungal infections are often indirect consequences of toxicologic insult. An additional challenge for many toxicological pathologists is the unfamiliarity of invertebrate gross and microscopic anatomy, and the morphologic responses of such organisms to chemical challenge. For example, the convoluted internal organization of cnidarians such as corals and anemones can be quite daunting to the uninitiated, not to mention the uncharacteristic appearance of many inflammatory and neoplastic lesions. However, it is likely that histopathology will become an increasingly utilized tool for investigating the toxicological fallout of pollution in fragile invertebrate populations, as exemplified by the apparent sensitivity of marine corals to the toxic effects of environmental contaminants such as oil dispersants.

Major Disease Models

In another example, it has become evident that sea urchin embryos (SUEs) are suitable models for developmental neurotoxicity in mammals, due to conservancy of brain neurotransmitters such as acetylcholine, serotonin, dopamine, and norepinephrine, which are utilized in the SUE as growth regulatory signals ( ). SUEs have also been used to investigate multidrug efflux transporter activity ( ). A further interesting model is the California sea hare, Aplysia californica , which was the first mollusk to be genomically sequenced. The central nervous system of Aplysia contains only 20,000 neurons, as compared to the 10 ¹² neurons present in mammals, and some of their largest neurons approach 1 mm in diameter, which makes them the largest somatic cells in animals. The size of Aplysia neurons provides several advantages for neurotoxicity research: these cells can be easily manipulated via dissection or injection, the behavioral functions of individual neurons can be mapped, and they can even be cultured for the purpose of generating in vitro neural networks ( ).

To date, one of the most convincing models of reproductive endocrine disruption is the occurrence of “imposex” in marine snails that have had environmental or experimental exposure to organotin compounds. The term “imposex” refers to a condition in which the male genitalia (penis, vas deferens) are irreversibly superimposed on the genitalia of female marine gastropods ( Figure 22.4 ). Exposure to tribultyltin (TBT), a biocide ingredient in antifouling paints applied to the undersides of ships and other marine installations, is considered to be the predominant cause of this particular reproductive tract malformation. There are numerous lines of evidence linking TBT to imposex formation, including the following: (1) the historical onset of imposex coincides with earliest utilization of TBT; (2) strong correlations exist between ambient concentrations of TBT and the prevalence and/or severity of imposex, and between the severity of imposex and tissue concentrations of TBT; (3) sex ratios are skewed to male and juvenile recruitment is reduced in TBT-impacted locations; (4) snails transplanted from clean sites to TBT-contaminated harbors absorb TBT and develop imposex; (5) the occurrence of imposex in laboratory-reared snails and those from pristine habitats is very low or nonexistent; and (6) snail populations have recovered from imposex and reemerged in at least some of the areas where TBT has been partially banned ( ). Several mechanisms have been proposed for imposex induction, including inhibition of testosterone excretion, interference with testosterone esterification, neuropeptide-like effects on genital development, inhibition of aromatase, and agonistic activation of the retinoid X receptor. Imposex frequency in certain prosobranch snails has been used as a biomonitoring tool for TBT contamination, facilitated by standardized systems of imposex severity measurement such as the vas deferens sequence index and the relative penis size index ( ).

Basic Biologic Characteristics and Common Species

As with the other nonmammalian animal species, the use of fish in toxicology varies widely, from field research in ecotoxicology to target animal safety (TAS) studies for aquaculture to laboratory-based chemical screening and basic research. Accordingly, the fish species used are widely diverse in terms of their biological characteristics. However, those species most commonly used in a laboratory setting typically offer the advantages of small size, high fecundity, and rapid external development of embryos ( ; ; ). While the zebrafish Danio rerio has become the all-around workhorse among fish species in biomedical research and drug discovery, several other fish species maintain their utility in laboratory toxicology investigations, particularly for endocrine disruption studies. Some of the most commonly used species include the Japanese medaka Oryzias latipes ( ) and the fathead minnow Pimephales promelas ( ; ) . The rainbow trout O. mykiss is used in endocrine disruption studies and is a frequently employed tumorigenesis model ( ).

Background Findings

While a detailed discussion of background findings in fish is beyond the scope of this chapter, it is worth reviewing features of certain key tissues (liver, gill, kidney, and gonads) pertinent to toxicologic investigations in fish. It should be noted that, as is the case in mammals, toxicologic effects in fish are not restricted to these tissues. Spontaneous neoplasms in fish will be briefly discussed as these may be encountered in ecotoxicological research or aquaculture.

The liver is the most frequent site for chemically induced primary tumors in fish, and in company with the gills and kidneys, it is a preferred target for noncarcinogenic toxicity. Compared to the mammalian liver, the fish liver is generally less sensitive to hepatotoxicants, and several anatomical and physiological factors may be responsible for relative differences in susceptibility ( ). For example, because fish hepatocytes are arranged as a system of blind-ended, anastomosing, and branching tubules rather than cords, exposure to toxicants may be reduced because sinusoidal perfusion is limited to only the basal and basolateral aspects of hepatocytes. Fish livers are also smaller than mammalian livers relative to body weight and they are 25%–50% less perfused; these factors may further limit toxicant exposure. Additionally, because hepatic lobules are not sharply defined and biotransformation enzymes tend to be homogenously distributed, toxic effects tend to occur diffusely, as opposed to the more concentrated centrilobular, midzonal, or periportal patterns of hepatocellular change that are often seen in mammalian livers. Physiologically, hepatic monooxygenase cytochrome enzymes such as CYP1A appear to be less consistently induced by xenobiotics in fish livers. However, fish livers are certainly susceptible to a variety of hepatotoxicants, including classic examples such as carbon tetrachloride, acetaminophen, and microcystins (blue-green algal toxins) ( ). Compared to mammals, the regenerative ability of fish livers following toxicant-induced damage is, in some cases, nothing short of phenomenal ( ).

As previously stated, the gills are another preferred site for toxic responses in fish, especially in terms of morphologic effects. Undoubtedly, the inherent vulnerability of the gill tissue to chemical trauma is due in part to the continuous exposure of the single-layered lamellar epithelium to the water environment. Additionally, the gills have a high level of metabolic activity and diverse physiological responsibilities, as they play major roles in respiratory gas exchange, ionic and acid–base balance, and the excretion of nitrogenous waste substances, and a contributing role in the extrahepatic biotransformation of xenobiotics ( ). A consequence of this multiplicity of physiological demands is that the gills provide numerous targets for waterborne toxins that include carrier-based ionic exchange mechanisms, paracellular pathways of fluid and ion exchange, molecular transport enzymes, and vascular hormone receptors ( ). Morphologic responses of the gills to toxic insults are varied but not infinite, and include the following: necrosis or desquamation of epithelial pavement cells (squamous cells covering the lamellar surface) or pillar cells (modified endothelial cells supporting gill lamellae); epithelial cell hyperplasia and hypertrophy; adhesion or fusion of (secondary) lamellae or filaments; lamellar edema; hemorrhage within filaments or lamellae; lamellar telangiectasis and/or thrombosis; increased leukocyte infiltration; changes in chloride cell numbers; increased rodlet cells; mucus cell hyperplasia; and excessive mucus production. Although patterns of histopathologically evident effects may suggest certain etiologies (for example, lamellar adhesions are often sequelae to metal toxicity or bacterial gill disease induced by Flavobacterium spp.), changes in the gills are most often nonspecific. Additionally, it may be difficult to differentiate cases of targeted gill toxicity from chemical “irritation” (e.g., caused by pH extremes) or osmotically induced stress, especially if the toxic effects become complicated by opportunistic bacterial or fungal infections, uncommon but potentially confounding factors in toxicity studies ( ; ; ). Nonneoplastic proliferative responses to chemically induced injury can occur with such rapidity in the gills that the term “chronic” is often not used as a temporal modifier in histopathologic diagnoses, and similar to the liver, the regenerative capacity of gill tissue can be remarkable if the fish survives the initial insult. Among the ever-expanding list of natural and man-made substances that cause gill toxicity in fish are metals (e.g., aluminum, copper, zinc, cadmium, cobalt, silver, and mercury), organochlorines, organophosphates and carbamates, herbicides, petroleum compounds, detergents, chemotherapeutic agents, and nitrogenous waste materials such as ammonia (for comprehensive lists, see , and ).

Anatomically and functionally, the fish kidney differs from the mammalian kidney in a number of respects. Adult fish have a mesonephric (= opisthonephric) kidney that is primitive relative to the metanephric kidney of mammals, and as such, it contains far fewer nephrons (10–50 as compared to approximately one million in higher vertebrates), and the renal parenchyma is not organized into distinct cortical and medullary regions. Nephrons generally lack a loop of Henle; consequently, most fish cannot increase the solute concentration of their urine. Reductionism is a trend in saltwater fishes (which are evolutionarily more advanced than freshwater fishes), and nephrons in some marine species lack distal tubular segments and/or glomeruli ( ). Blood flow to the fish kidney approaches that of the entire cardiac output due to the existence of a renal portal system ( ). Structural elements of the hemopoietic, immunologic, and endocrine systems are located in the kidney, usually in the anterior portion; therefore, in addition to supplementing the gills in the elimination of nitrogenous wastes, the fish kidney also participates in activities as diverse as hematopoiesis, lymphopoiesis, antigen processing, calcium regulation, and the production of corticosteroid and adrenergic hormones. From a toxicologic standpoint, the excretory kidney and the interstitial hematopoietic/lymphopoietic tissue are probably the two most common targets for microscopically visible effects. Consequences of toxicologic insult to the fish nephron are qualitatively similar to the types of effects that occur in mammals, and commonly observed changes include the following: renal tubular vacuolation; degeneration and necrosis; thickening of glomerular membranes and mesangium; dilation of glomerular capillary loops; glomerular vascular thrombosis; alterations in the size of Bowman's space; periglomerular fibrosis; and secondary interstitial inflammation. As in mammals, the proximal tubule is particularly susceptible to injury as it bears primary responsibility for the renal excretion of xenobiotics and their metabolites ( ). There is evidence that proximal tubule damage may be at least partially mediated by endothelin-induced decreases in the multidrug resistance protein 2–associated transport of toxic substances into the urine ( ). The adult fish kidney has the ability to wholly regenerate nephrons following injury, as demonstrated, for example, in a study in which goldfish were exposed experimentally to the nephrotoxic compound hexachlorobutadiene ( ). It is perhaps because of this regenerative ability, and the fact that the gills are the primary site for the elimination of nitrogenous wastes, that fish appear able to tolerate a proportionally greater amount of renal tissue damage as compared to mammals. Additional substances known to be nephrotoxic in fish include metals such as cadmium (for which the kidney is the primary target organ) and mercury; organophosphate, carbamate, and organochlorine pesticides; and aminoglycoside antibiotics ( ). Effects suggestive of immunotoxicity and/or impaired hematopoiesis have been observed in the renal hematopoietic tissue. Substances considered to be immunotoxic in fish include PAHs, halogenated aromatic hydrocarbons, metals, and organometals ( ). In several fish species, the administration of high doses of certain antibiotics (e.g., oxytetracycline, florfenicol) has been associated with decreases in numbers of lymphopoietic and/or hematopoietic cells located in the interstitium of the anterior kidney, with or without overt evidence of individual cell necrosis or apoptosis ( ). Expansion in the size and/or number of pigmented macrophage aggregates (clusters of constituent histiocytic phagocytes that function as reservoirs for cell breakdown products and as sites of antigen presentation) in the kidney, and other tissues, has also been used as a marker of prior toxicologic insult ( ).

Histopathology can be a valuable tool in reproductive endocrine disruption compound (EDC) studies involving fish because piscine gonads are quite labile during early development and even in the adults of some gonochoristic species in which the gonadal sex does not normally change during the animal's lifespan. In addition to intersex (the presence of opposite sex tissue in the gonads), characteristic microscopic changes in the gonads in response to EDCs can include the following: malformations of the gonadal efferent duct system; variations in the proportions of different gametogenic precursors (e.g., estrogenic exposure has been associated with increased spermatogonia relative to other cell stages); altered gametocyte production; gametocyte degeneration (e.g., excessive or inappropriate ovarian follicular atresia); decreased yolk formation in ovarian follicles (a consequence of aromatase inhibitors); and the increased prominence of somatic cells such as Leydig (interstitial) cells in testes or granulosa cells in ovaries. Histopathologic changes caused by the exogenous administration of reproductive hormones or their anthropogenic mimics are not restricted to the gonads. During periods prior to spawning, the livers of reproductively mature female fish typically generate a phospholipoglycoprotein called vitellogenin that is a required ingredient for egg yolk production. Vitellogenesis is associated with an upregulation of organelles (e.g., rough endoplasmic reticulum) in hepatocytes that often imparts a noticeable basophilic coloration to the cytoplasm of the liver cells in hematoxylin-stained histologic specimens. Excessive amounts of vitellogenin protein in the plasma of fish exposed to potent estrogenic substances, for example, can be visualized in histologic sections as dark pink homogenous material within the vasculature at various systemic sites. Furthermore, in male fish (which normally have minimal measurable circulating vitellogenin), chemically induced production of vitellogenin by the liver has been linked to profound renal glomerular and tubular damage ( Figure 22.5 ), which is presumably caused by protein overload of the renal excretory system ( ).

Although the background incidence of neoplasia in most fishes tends to be low, many pathologists may be surprised to learn that most of the same types of epithelial, mesenchymal, and round cell tumors found in mammals have been observed to occur spontaneously in fish, and the histomorphology of many preneoplastic and neoplastic proliferative lesions is often strikingly familiar. Distal or localized spread of malignant neoplasms occurs less commonly in fish than in mammals, but when tumor tissue does travel, it often localizes in the liver ( Figure 22.6 ). Species predilections exist ( Table 22.3 ) and, in some, oncogenic viruses have been shown or suspected to cause neoplasms ( ; ; ). Spatial associations have been recognized between the presence of some other types of infectious organisms and the formation of hyperplastic lesions and tumors, and examples include the cooccurrence of pseudocapillaria nematodes and intestinal and ductal (pancreatic or biliary) carcinomas in zebrafish ( ), and microsporidian organisms and ovarian granulosa cell tumors in longjaw mudsuckers Gillichthys mirabilis ( ). However, despite the recognized ability of viruses, parasites, and various carcinogenic chemicals to cause cancer in fish, at least by experimental exposure, for most naturally occurring piscine neoplasms, the reasons for tumor formation are unknown.

Major Disease Models

The earliest known toxicological tests in fish primarily involved acute studies and lethal outcomes, in which one of several common species of freshwater carps, minnows, or salmonids was exposed to anthropogenic compounds to determine the relative toxicity of various substances found in natural waterways. During the last several decades of the 20th century, however, scientists began to fully recognize and appreciate the ability of fish to form a variety of proliferative lesions in apparent response to environmental chemical exposure. In the United States, field investigations of certain nonmigratory and benthic fishes that inhabit coastal waters known to be contaminated by industrial or sewage effluents provided seminal results ( ). In impacted Pacific coast sites such as Puget Sound, Washington, San Francisco Bay, and others, the discovery of neoplastic and preneoplastic liver lesions in bottom-dwelling fishes such as the English sole Pleuronectes vetulus and starry flounder Platichthys stellatus , and the potential association between the prevalence of these lesions and polychlorinated biphenyls (PCBs), PAHs, and other nonnatural contaminants prompted US Congressional hearings and legislative activity ( ). Further impetus for toxicologic research was provided by the decades-old discovery of contaminant-associated liver and pancreatic neoplasms in mummichogs F. heteroclitus (an estuarine killifish) in the Elizabeth River, Virginia, USA ( ), and liver and dermal tumors in brown bullhead catfish Ameiurus nebulosus in the Great Lakes, its tributary rivers, and other Midwestern and Northeastern US waterways ( ). The impact that these discoveries, and those of other fish tumor epizootics, have had on environmental research can hardly be overstated. In fact, fish from many of these locations are still being monitored for tumor burden to document the effects of contaminant site remediation, and reductions in the concentrations of carcinogens in the water, sediment, and fish tissues have been associated with declines in tumor incidence at some impacted sites ( ).

The emphasis of environmental research in fish over time has shifted gradually from studies that primarily involved known or suspected carcinogens and heavy metals to those focused on various agricultural, industrial, and pharmaceutical chemicals that have been associated, at least circumstantially, with nonneoplastic toxicity and endocrine disruption. As with invertebrates, toxicity testing in fish has become a critical part of environmental impact assessments required for certain new drug applications ( ; ) and a mainstay in EDC studies ( ). Some of the most widely used fish species in EDC experiments include Japanese medaka O. latipes , zebrafish D. rerio , fathead minnow P. promelas , three-spined stickleback Gasterosteus aculeatus , guppy Poecilia reticulata , sheepshead minnow Cyprinodon variegatus , and rainbow trout O. mykiss . Each of these species has its own intrinsic benefits and drawbacks as a model organism for EDC studies. For example, one advantage of using fathead minnows is that reproductively active males develop macroscopically evident, external secondary sex characteristics that include specialized coloration, nuptial tubercles (a bilaterally symmetrical pattern of small, wart-like protuberances on the face), and enlargement of the dorsal nape pad (a subcutaneous deposition of loose collagenous tissue along the dorsal aspects of the head and neck). The development of these features is androgen dependent, and conversely, their phenotypic expression can be reduced or eliminated in a dose-dependent manner by exposure of the fish to exogenous estrogens or substances with estrogen-like activity ( ). Alternatively, a different set of advantages is offered by use of the three-spined stickleback model. Male sticklebacks produce a glue-like protein in their kidneys called spiggin that is essential for nest building, and spiggin production is under the control of androgens. Female sticklebacks normally do not produce this protein; consequently, the induction of measurable concentrations of renal spiggin in female sticklebacks that were exposed experimentally to contaminated effluents has been used as a biomarker for exogenous androgenic activity ( ). In a similar fashion, the potential antiandrogenic activity of xenobiotic substances can be determined by the extent to which they inhibit spiggin production in males ( ). A further advantage of using G. aculeatus is the existence of DNA markers that can be used to determine the genotypic sex of individual fish ( ). Because most fish species do not have heteromorphic sex chromosomes, the existence of genetic markers for sex is a plus for experiments in which exogenous hormone administration results in an intersex condition, i.e., the genetic sex can be determined in instances where the phenotypic sex is ambiguous. Thus, the results of nonmorphologic endpoints such as these can aid the pathologist by providing context for understanding the relevance and potential mechanism(s) of action associated with certain pathologic findings.

Currently, any in-depth discussion of nonmammalian animals used in toxicological bioassays and drug screening would have to acknowledge the dominance of the zebrafish as a biomedical research model. A contemporary testament to the importance of this model, in addition to the mountain of published work that has accumulated over the past decade, is the ubiquitous presence of zebrafish colonies in the laboratories of universities, human medical centers, pharmaceutical companies, and the research arms of government agencies across the globe. Some of the medical research areas in which zebrafish have made the greatest contribution, or shown the most promise, include embryogenesis, immunology, hemostasis, cardiology, functional anatomy of ocular and auditory organs, endocrine disruption, developmental neurology, angiogenesis, oncology, and aging. As experimental subjects, zebrafish possess all of the advantages that are typically associated with other species of small ornamental aquarium fish, such as high fecundity, short generation time, ease of culture, hardiness, small volume dosing, the ability to administer test compounds via water bath exposure, the capacity to house large numbers of animals in a small space, and the capability of viewing multiple organ systems in relatively few histologic sections. However, zebrafish have a number of bonus attributes that have caused them to vault past other fishes in terms of research utility. Consequently, they have become a universal model for many types of toxicologic studies, including those involving basic biomedical research, drug development and screening, toxicity testing (acute, developmental, or organ specific), and ecotoxicologic investigations ( ). Zebrafish spawn year round and are oviparous (the eggs are scattered and externally fertilized), and the unparalleled transparency of their early stage embryos allows visualization of several rapidly emerging organ systems, full development of which occurs by 96 h postfertilization, which is roughly equivalent to that of a 3-month-old human embryo. The period of transparency can be extended by adding the tyrosinase inhibitor 1-phenyl-2-thiourea to the growth medium, or alternatively, the casper mutant strain of apigmented zebrafish can be utilized ( ). Transparency also facilitates a wide variety of manipulations that involve, for example, microinjections of nucleic acid materials or the microsurgical removal or transplantation of specific embryonic cell lines. A vast array of mutant strains is available ( ; ). Some of these manipulated strains have been found to mimic genetically based human diseases, not only molecularly but also phenotypically in some cases, whereas other transgenic zebrafish are employed in studies of gene function, efficacy screening of pharmaceutical compounds, and toxicity testing ( ). The fact that zebrafish embryos can be reared in 96-well plates for up to 5 days, without requiring supplemental feeding due to their attached yolk sacs, allows them to be used in whole-animal high-throughput assays for screening drugs or toxic chemicals. Imaging platforms specifically designed for these zebrafish embryo setups facilitate rapid, automated evaluation of in vivo morphologic endpoints relevant to cardiotoxicity and neurotoxicity, among others. In one of the most efficient uses of such assays, anesthetized transgenic zebrafish embryos that expressed a green fluorescent protein (GFP) marker were combined with automated imaging and analysis to screen for compounds that modulated the FGF/RAS/MAPK intracellular signaling pathway ( ).

Similar to laboratory rodents, there are several strains of zebrafish commonly in use. Mapping of the zebrafish genome has been completed for the Tuebingen strain. An annotated assembly of this reference genome is maintained and periodically updated ( https://www.ncbi.nlm.nih.gov/grc/zebrafish ), facilitating comparative genomics and other genetic-based research. Studies to characterize strain differences and their potential impacts on research are ongoing.

Basic Biologic Characteristics and Common Species

Amphibians, especially frogs, are excellent subjects for toxicological studies. Frog experiments can be conducted in the laboratory, in the field, or in intermediate enclosed systems known as “mesocosms,” which offer advantages of controlled experiments with “real-world” variables. Not only are they small animals with short generational times, their aquatic phases can be exposed to test agents continuously via water bath, and exposure can occur simultaneously through multiple absorption routes (i.e., via the gills, skin, and gastrointestinal tract). Amphibians typically have complex life cycles in which the juvenile stages (egg, embryo, and larva) are particularly sensitive to a variety of toxicants, and chemically induced disturbances in these developing animals often produce dramatic morphologic changes. The potential for exposure to environmental contaminants is very high in amphibians for a number of reasons: they lay permeable eggs in water; their skin is permeable to fluids and gases; most have both aquatic and terrestrial phases in their life cycle, plus a physiologically demanding metamorphic period; the algal diet of young amphibians and carnivorous diet of adults enhance the opportunity to ingest contaminated food; and they hibernate in sediment, which is a well-documented repository for a variety of anthropogenic pollutants ( ). The elaborate life cycles and well-characterized behavioral traits of frogs ensure that a wealth of endpoints, some universal and others uniquely amphibian (e.g., hind limb resorption), are available for toxicologic bioassays. Tumor development, whether spontaneous, virally induced (e.g., Lucke's herpesvirus [ranid herpesvirus 1]), or caused by other factors, occurs in all major organ systems of both anurans (frogs and toads) and urodeles (tailed amphibians) ( ); in general, responses to chemical carcinogens in amphibians tend to parallel those of mammals.

Based on recent literature reviews ( ; ), the most utilized amphibians for contaminant-based research are members of the Ranidae (true frogs), followed by the tongueless Xenopus spp. African clawed frogs, bufonids (true toads), caudate (urodele) amphibians (salamanders and newts), and hylids (tree frogs). Although it is often desirable to simulate authentic ecotoxicological conditions by using amphibians that are native to a particular region of interest, native species can be challenging research subjects for a number of reasons: they may only spawn seasonally and they often have low laboratory survival; it may be difficult to capture adequate numbers of specimens for testing; standardized protocols may not be available for certain species; and collection of animals in the wild may place added pressure on at-risk populations. Presently, the premier amphibians for many types of laboratory research are Xenopus spp. frogs, including, in particular, Xenopus laevis , and the closely related Xenopus ( Silurana ) tropicalis . Unlike many other amphibians that have terrestrial phases, defined breeding seasons, and require several years of development to attain sexual maturity, laboratory-cultured Xenopus frogs are entirely aquatic, they can be induced to spawn year round, and they attain reproductive maturity in 1–2 years or less, depending on the species ( ). Additionally, Xenopus has long been a preferred model for early developmental and gene expression studies due to the large size and sturdiness of their embryonic cells and eggs, and the amenability of their external embryos to surgical manipulation. Although X. laevis is by far the most studied of the 19 members in the genus, the smaller X. tropicalis has advantages that include a more rapid generation time and the ability for researchers to determine the genotypic sex of individual frogs ( ). While polyploid genomes are common among many amphibian species ( ), X. tropicalis offers the additional advantage of a diploid genome that is also fully sequenced ( https://uswest.ensembl.org/Xenopus_tropicalis/Info/Index?db=core ).In terms of ecotoxicological experiments, the major criticism of Xenopus as a surrogate test animal is that they are behaviorally, anatomically, and physiologically different from many native amphibians, and as a result, may not exhibit comparable sensitivity to toxicants. Despite that alleged shortcoming, Xenopus has been employed as a laboratory subject for research disciplines as diverse as tissue regeneration, drug screening, tumorigenesis, immunology, and thyroid and reproductive endocrine disruption. These frogs are now continuously available through commercial sources, and decades of research efforts have culminated in the production of various X. tropicalis transformants through the use of a variety of elegant transgenesis procedures ( ). Standardized criteria developed for the visual staging of Xenopus embryologic development ( ) have allowed investigators to assess the effects of toxicants on the timing and completeness of amphibian metamorphosis, which is a process that is known to be highly thyroid hormone dependent ( Figure 22.7 ).