Toxicologic Pathology

The field of toxicologic pathology is being impacted, as are other fields of science and medicine, by rapid transitions to take advantage of new science and technology. The new technology represents great opportunities to advance our understanding of toxicology and pathology to exciting new levels, but it also poses new challenges. We must be seriously engaged in that transition to assure that the outcome reflects the knowledge and discipline that are hallmarks of today's decision-making process in areas of product development and approval. New expertise will be required to deal with new issues. How well and how rapidly we adapt as the field moves from “... icities” to “ ... omics” will, at least in part, determine the role of toxicologic pathologists in the product development and approval processes of the future.

Interpreting canine and nonhuman primate clinical pathology data from preclinical studies can be challenging. Relatively few animals are tested (typically beagles and macaques), and they often undergo study-related procedures (eg, sample collection for pharmacokinetic analysis) that can affect clinical pathology test results. Data interpretation requires an understanding of the significance of each test, species differences for each test, normal interanimal and intraanimal variability, the effects of study design variables, and supporting data from other disciplines. Interpretation of hematology, coagulation, clinical chemistry, and urinalysis parameters are discussed, with emphasis on species peculiarities and study design variables that may affect clinical pathology test results.

During the development of a new therapeutic, few pharmacodyamic outcomes currently receive as much scrutiny as the effect of a potential medication on the electrocardiographic QT interval. The recent withdrawal from marketing of several drugs due to potential drug-related cardiac arrhythmias have greatly increased concern about drug-related changes on the QT interval. In order to reduce the incidence of these idiosyncratic episodes, regulatory agencies have suggested that sponsors use more rigorous methodology during the safety evaluation of new pharmaceuticals. Along with enhanced electrocardiographic assessments during clinical trials, advanced preclinical examinations of effect on QT interval and ventricular repolarization have become de rigueur. In this arena, the beagle dog is the preclinical species often associated with the most reliable predictivity for human safety assessment. To this end, canine models of cardiovascular safety assessment are discussed along with the relevance of these assays to human electrocardiography.

The dog is a commonly used animal model by virtue of its size, well-characterized physiology, and ease of handling. For these reasons and others, dogs are also useful in pharmacokinetic and metabolism studies during the development of both human and veterinary pharmaceutical products. In comparison with humans, or with other animals, dogs have some unique physiologic attributes that can affect the disposition of drugs. Species differences in gastrointestinal physiology, metabolism, renal function, and protein binding can affect the correlation of the pharmacokinetics and toxicology of dogs with those of other species. With the use of relevant examples, this article will provide an introduction to characteristics of dog physiology and their impact on pharmacokinetics, metabolism, drug disposition, toxicity, and dose selection.

When vascular injury is observed in dogs used in preclinical toxicology studies, careful evaluation of the lesions is warranted, especially when differentiating drug-induced vascular changes from spontaneous findings, such as idiopathic canine polyarteritis. The clinical signs as well as the nature and distribution of lesions can often be distinguishing, as is the case with vasoactive drugs, including vasodilators and/or positive inotropes (hydralazine, minoxidil, endothelin receptor antagonists, and phosphodiesterase III inhibitors). For most types of vasodilator-induced vascular injury, the lesion is often restricted to coronary arteries, whereas in idiopathic canine polyarteritis, arterial lesions not only involve coronary arteries, but also medium to small arteries of other organs. In addition, the nature of the changes in vessels yields important clues. Medial and adventitial hemorrhage is generally associated with vasodilator-induced arterial lesion, whereas hemorrhage is generally absent in idiopathic polyarteritis. Although idiopathic canine polyarteritis can generally be differentiated from vasoactive-induced vascular injury in dogs, there are increasing incidences of this type of polyarteritis in dogs receiving any 1 of a number of unrelated classes of compounds, suggestive of an exacerbation of the spontaneous disease. Therefore, in order to differentiate drug-induced injury from idiopathic canine polyarteritis, it is critical that examination of the vascular pathology be conducted with good understanding of clinical, pharmacological, and mechanistic data associated with the drug.

Sampling of large-sized brains (eg, dog, primate) for microscopic examination is frequently inadequate to detect localized neurotoxic injury. Furthermore, the examination of H&E-stained sections alone will often be insufficient for the detection of subtle neuropathogic alteration. It is imperative for any pathologist evaluating brain sections to have knowledge of microscopic neuroanatomy and to also have some understanding of basic neurochemistry. When a focus of degeneration is detected within the brain, the pathologist needs to ascertain not only the specific anatomic location of this focus but also the neuroanatomic regions that project to and receive output from the injured focus. Because of the complexity of brain circuitry and the fact that the brain contains many distinctive neuron populations, many more brain sections are required for adequate microscopic evaluation than for any other body organ. Deciding which and how many areas should be examined, microscopically, from a large size brain is often problematic. Although any sampling protocol will be influenced by what is known about the test chemical, it has been well established that certain regions of the brain (eg, hippocampus and other components of the limbic system, basal ganglia, Purkinje neurons) are more susceptible than others to a variety of physical, metabolic, and chemical insults. Knowledge of these regional sensitivities will assist in guiding the pathologist in the development of an adequate sampling protocol.

In the context of human hematopathology, the terms myelodysplasia and Myelodysplastic Syndromes (MDS) are applied to disorders of hematopoiesis that are clonal, neoplastic, and, in most forms, manifested as ineffective hematopoiesis with characteristic morphologic abnormalities in multiple cell lines. Studies of human patients have provided the conceptual framework that MDS evolve from a multipotential hematopoietic stem cell (CFU-GEMM) that has undergone neoplastic transformation as the result of acquired genetic mutations. The diagnosis of MDS in human patients is based largely on morphologic examination of marrow but can be confirmed by detection of cytogenetic abnormalities. Spontaneous, neoplastic myelodysplasia occurs in dogs, but rarely. Nonneoplastic syndromes of ineffective hematopoiesis are more common in dogs, can resemble MDS, and are probably immune mediated in many cases. Drugs and their metabolites are potential causes of dysmorphic maturation and ineffective hematopoiesis. Without methods to confirm clonality by cytogenetic analysis, the diagnosis of neoplastic myelodysplasia in dogs is based on light microscopic examination of bone marrow smears. This paper discusses and illustrates the characteristic morphologic and cytochemical features of neoplastic myelodysplasia and nonneoplastic ineffective hematopoiesis in dogs.

Ready or not, fish models are “here to stay.” No longer are fish confined to a few specialized laboratories, nor are they exclusively the purview of zoologists or environmental toxicologists. In fact, the institution that does not house at least 1 fish facility is probably not at the forefront of cutting edge research. In toxicologic pathology, fish models are increasingly being used to provide high animal numbers at relatively low cost in carcinogenicity testing and developmental research, and to provide mechanistic information on fundamental cellular processes. In this session, we attempt to provide some perspective for the pathologist that is faced with planning or performing experiments or testing protocols using fish models, or with reading or interpreting fish studies. First, we cover how to approach fish studies from the contract laboratory standpoint, including sectioning, quality control, and GLP considerations. Then, we discuss specifics on the use of the rainbow trout, zebrafish, and Japanese medaka models. The rainbow trout has a rich history in carcinogenicity and mechanistic cancer research. Similarly, the 2 workhorses in the small fish category, zebrafish and medaka, have found their way into many laboratories doing developmental biology and genomics research as well as carcinogenicity testing. Some fascinating genetically altered fish models have been developed with both of these species. This manuscript provides a session overview of the use of small fish models in toxicologic pathology, along with some historical perspective on how these models have played a role in the current state of the science.

In the late 1970's, Good Laboratory Practice Regulations (GLP) were instituted by agencies such as the USFDA, the USEPA, and the OECD to provide a system for the monitoring of animal studies submitted in support of the safety of regulated products. Although GLP regulations are regularly employed in laboratory mammal projects, they have been comparatively under-utilized in aquatic animal research. This situation is changing due to the continuing emergence of fish as toxicological and pharmaceutical test subjects, human and animal disease models, genetically-engineered food sources, and environmental sentinels. The application of GLP principles to aquatic animal studies poses a variety of challenges, especially in the areas of Study Protocol design and the creation of Standard Operating Procedures (SOP's). This presentation will highlight differences between mammalian and fish studies in the application of GLP principles, and identify specific concerns associated with the formulation of SOP's for fish projects.

The rainbow trout has been utilized as a model for human carcinogenesis for a number of years. Trout are relatively inexpensive to maintain and exhibit (over the 9—12-month tumor assay period) very low spontaneous tumor backgrounds. One of the most powerful applications of this model is the design and conduct of large-scale tumor studies requiring thousands of animals that address statistically challenging questions of dose-response. Two recent examples of such applications include our studies on I3C as a tumor promoter and DBP as a tumor initiator. I3C was shown to promote AFB1- initiated liver cancer at doses near those recommended for supplementation in humans. Further studies are required to determine if the mechanisms responsible for promotion in trout can be extrapolated to humans. In the second example, we report results from the largest animal tumor study ever conducted. A total of 42,000 trout were utilized to measure DBP carcinogenesis down to incidences of 1 in 5,000. The dose response model deviated significantly from linearity although the existence of a threshold could not be statistically established. Extrapolation of the data model predicts a DBP dose producing 1 in 106 cancers that is 1,000-fold higher than predicted by the conservative linear model. If these results can be confirmed with other carcinogens (genotoxic and perhaps nongenotoxic) and other targets, this could have a significant impact on the utilization of animal tumor data in human risk assessment.

The zebrafish (Danio rerio ) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. The zebrafish genome will be completely sequenced within the next 1—2 years. Together with the substantial historical database regarding basic developmental biology, toxicology, and gene transfer, the rich foundation of molecular genetic and genomic data makes zebrafish a powerful model system for clarifying mechanisms in toxicity. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, our database regarding infectious and noninfectious diseases and pathologic lesions in zebrafish lags far behind the information available on most other domestic mammalian and avian species, particularly rodents. Currently, minimal data are available regarding spontaneous neoplasm rates or spontaneous aging lesions in any of the commonly used wild-type or mutant lines of zebrafish. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding human development, disease, and toxicology we must greatly advance our knowledge on zebrafish diseases and pathology.

A need exists for whole animal toxicity, mutagenesis, and carcinogenesis models that are alternative to the traditional rodent test models and that are economical, sensitive, and scientifically acceptable. Among small fish models, the Japanese medaka (Oryzias latipes) is preeminent for investigating effects of carcinogenic and/or toxic waterborne hazards to humans. The guppy (Poecilia reticulata ), although less widely used, is valuable as a comparison species. Both species are easy to maintain and handle in the laboratory and there is a large body of background information on their responsiveness to a range of classes of carcinogens. There are considerable data on the occurrence of background diseases and on spontaneous neoplastic lesions, both of which occur relatively rarely. With few modifications, the medaka and guppy are amenable to carcinogenicity testing under the rigid standards established by the National Toxicology Program (NTP) for rodent tests. The advantages of the small fish in carcinogenesis studies are best realized in long-term studies that involve environmentally realistic exposures. Studies to identify chronic effects can be conducted in about 12 months, near the life span of medaka in our laboratory. Practically, 9-month studies are optimal but shorter study cycles and a variety of exposure/growout and initiation/promotion scenarios are available. Studies on 3 compounds tested in medaka under NTP protocols are under review and preliminary analysis indicates that chronic carcinogenicity bioassays with medaka, guppy, and potentially with other small fish species are feasible and scientifically valid.

Nonhuman primates are important laboratory animals for biomedical, pharmacology, and toxicology research. To effectively use primates as models, their gross and histologic anatomy, physiology and natural history, as well as common health problems and the source from which the primate is obtained, must be known and understood by pathologists involved in study design and/or interpretation. The first very important lesson in the “primer” is: there is no such thing as a generic monkey. Brand names (ie, species and subspecies) are important. Several taxonomic groups of primates are used in research including: prosimians, such as galagos and lemurs; New World monkeys, particularily marmosets; Old World monkeys, especially macaques and baboons; and the chimpanzee, an African ape. Differences between taxa are exemplified by the glucocorticoid resistance of New World monkeys compared to Old World monkeys, which results in the requirement for Vitamin D3 and their high circulating levels of steroids such as cortisone and progesterone. Differences in ovarian histology between Old and New World monkeys probably relate to steroid receptor biology as well. There are also variations in disease manifestations, even among closely related primate species such as rhesus and cynomolgus macaques (cynos). For example type D retrovirus infection is accompanied by lymphomas in cynos, but not rhesus. The second important lesson in this “primer” is: “not test article related” does not always mean “normal.” Lymphoid nodules in bone marrow or salivary gland, a common background finding in macaques, often signal the presence of type D retrovirus. Other histologic changes and normal anatomic variations may be confusing to individuals not routinely examining primate tissues. The objective of this paper is to familiarize pathologists with the use of primates in research as well as lesions and nonlesions (normal anatomy or physiology) of primates that may influence study design and confound interpretation.

Various species of nonhuman primates are natural hosts for 6 exogenous retroviruses, including gibbon-ape leukemia virus (GaLV), simian sarcoma virus, simian T-lymphotropic virus (STLV), simian immunodeficiency virus (SIV), simian type D retrovirus (SRV), and simian foamy virus (SFV). These viruses establish persistent infections with a broad spectrum of pathogenic potential, ranging from highly pathogenic to nonpathogenic, depending on various host, virus, and environmental factors. Latent or subclinical infections are common, and various procedures associated with experimental protocols may lead to virus reactivation and disease. Adverse effects on toxicologic research by undetected retroviral infections can occur in several ways, including loss of experimental subjects (and statistical power) due to increased morbidity and mortality. In addition, results may be confounded by virus-induced clinical abnormalities, histologic lesions, alteration of physiologic parameters and responses, and interference with in vitro assays and/or destruction of primary cell cultures. Key clinical and epidemiological features of several important retroviruses are reviewed, with emphasis on viruses infecting species of macaques most commonly used as research subjects in primate toxicology studies. Examples of actual and potential confounding of toxicologic studies by retroviruses are discussed, including altered cytokine profiles in healthy STLV carriers, and clinical and pathological abnormalities induced by SRV infection. Adequate prestudy viral screening is critical to exclude retrovirus-infected primates from toxicologic research protocols and prevent potential confounding of research results.

Exposure to natural environmental products, biopharmaceuticals, or investigational adjuvants has the potential to negatively impact the immune system, resulting in either up- or downregulation of immune function (immunomodulation). Many current protocols for primate toxicologic testing call for the evaluation of changes in immune cell number (peripheral blood or tissue), alterations in the weights of immune system organs (lymph nodes, spleen, thymus), and/or increases in the overall incidence of infections or neoplasms; these data are relied upon to suggest altered immune function. However, these are informative only when clear differences in frequency and/or severity of effects can be distinguished across control and dosed groups. In the absence of such distinct morphologic or clinical pathologic changes, the identification of potential immunomodulatory effects can present a much greater challenge. Additional evaluations may be needed to detect altered immune system integrity; these are based on in vivo assessments in primates of cellular or humoral responsiveness. Immunomodulatory effects can be characterized by in vitro or in vivo immune function tests; these tests require prestudy planning to integrate assessments into ongoing toxicology programs. These methods also involve specialized training and equipment, particularly if the intent is to evaluate parameters in a GLP laboratory setting. In primate toxicology, the added costs required to perform a complete functional analysis of the immune system can be substantial, but may be warranted depending on the clinical development plans. Two analytical methods that are easily incorporated into the standard toxicology profile in primates are flow cytometry and immunohistochemistry. Flow cytometry (FC) is used to assess changes in the relative distribution of immune cell marker expression, and where marker expression is known to fluctuate with the state of cell activation, can also provide information on functional attributes of immune cells. Immunohistochemistry (IHC) provides a means to evaluate similar characteristics of immune cells within tissue sections. Used together, FC and IHC can aid in the identification of changes in immune system that may not be apparent by traditional testing procedures (such as H&E staining), thus aiding in the characterization of immune system alterations. This presentation focused on the utility of flow cytometry and immunohistochemistry in a standard primate toxicology evaluation, with representative examples showing the benefits of these technologies in the diagnosis of potential immunomodulatory effects.

Antisense oligodeoxynucleotide compounds (AS ODN) are being developed as therapeutics for various disease indications. Their safety and pharmacokinetics are most commonly evaluated in rodents and nonhuman primates. Traditional AS ODN are short, single strands of DNA, and they target specific mRNA sequences. Plasma clearance of AS ODN is rapid, broad tissue distribution occurs, and elimination is by nuclease metabolism. Structural modifications to AS ODN have been made to enhance their efficacy and improve their safety. A number of class effects are observed with AS ODN that are unrelated to the specific targeted mRNA sequence. Acute effects include activation of the alternative complement pathway and inhibition of the intrinsic coagulation pathway. In monkeys, rodents, and dogs given repeated doses of AS ODN, accumulation of AS ODN and/or metabolites occurs in the form of basophilic granules in various tissues, including the kidney, lymph nodes and liver. A new potential therapeutic application of ODN is that of immune stimulation. Immunostimulatory ODN (IS ODN) are being investigated for use in treating cancer, infectious disease, and allergy. For the development of both AS and IS ODN, primates will continue to be important for safety assessment.

The common marmoset, Callithrix jacchus, is the smallest nonhuman primate commonly used in biomedical research. Marmoset characteristics and propensities have enabled them to be used in a wide range of research as a model of human disease, physiology, drug metabolism, general toxicology, and reproductive biology. This paper provides a general overview of the marmoset with special emphasis on the benefits and disadvantages of this species as a model for inclusion in preclinical drug development programmes. In view of its small size in comparison with other nonrodent species marmosets have become of value for toxicology studies with biotechnology products where compound supply is limited. In general toxicology studies, marmosets have been successfully used to meet regulatory endpoints also for specific investigatory purposes. The widespread use of this species has allowed extensive background information to become available and a summary of the most frequently measured parameters are presented. Marmosets apparently represent an interesting animal model for comparative research on primate reproductive physiology. However, several basic aspects of reproductive processes exhibit cardinal discrepancies to those described for macaques and human. Thus, from the viewpoint of reproductive toxicology, the relevance of the marmoset primate model for human reproduction remains unclear to date and further research is obviously needed. Given our current knowledge of marmoset reproductive features, the use of this animal model cannot be recommended for reproductive toxicology assessment.

The issue of animal treatment has emerged as a major social concern over the past three decades. This ramified in a new ethic for animal treatment that goes beyond concern about cruelty and attempts to eliminate animal pain and suffering, whatever its source. This is evidenced by laws governing animal research in many countries. Insofar as toxicology can entail significant and prolonged animal suffering, it is at loggerheads with this new ethic. Ways are suggested for the toxicological community to put itself in harmony with the ethic and thereby preserve its autonomy.

The increasing emphasis on the provision of environmental enrichment to laboratory animals, vis-à-vis the USDA Animal Welfare Regulations, the Guide for the Care and Use of Laboratory Animals (NRC 1996), and a potential forthcoming policy from the USDA on the subject, can be difficult to accommodate in a toxicology research environment. A summary will be provided of current requirements and recommendations. Then, strategies for meeting regulatory requirements will be described for non-rodent animals used in toxicology research. These strategies will address methods of both social enrichment, such as pair or group housing, as well as non-social enrichment, such as cage furniture, food enrichments, and toys. In addition, the value of positive interactions with staff (e.g., through training paradigms or socialization programs) will also be discussed. Apparent in the discussion of these strategies will be an overarching recognition of the necessity to avoid introducing confounding variables into the research project and to avoid compromising animal health. The roles of the Institutional Animal Care and Use Committee (IACUC) and the attending veterinarian in helping scientists balance animal well-being, the scientific enterprise and the regulatory environment will be described.