The emerging field of translational safety genetics is providing new opportunities to enhance drug discovery and development. Genetic variation in therapeutic drug targets, off-target interactors and relevant drug metabolism/disposition pathways can contribute to diverse drug pharmacologic and toxicologic responses between different animal species, strains and geographic origins. Recent advances in the sequencing of rodent, canine, nonhuman primate, and minipig genomes have dramatically improved the ability to select the most appropriate animal species for preclinical drug toxicity studies based on genotypic characterization of drug targets/pathways and drug metabolism and/or disposition, thus avoiding inconclusive or misleading animal studies, consistent with the principles of the 3Rs (replacement, reduction and refinement). The genetic background of individual animals should also be taken into consideration when interpreting phenotypic outcomes from toxicity studies and susceptibilities to spontaneous safety-relevant background findings.
Gut homeostasis plays an important role in maintaining animal and human health. The disruption of gut homeostasis has been shown to be associated with multiple diseases. The mutually beneficial relationship between the gut microbiota and the host has been demonstrated to maintain homeostasis of the mucosal immunity and preserve the integrity of the gut epithelial barrier. Currently, rapid progress in the understanding of the host–microbial interaction has redefined toxicological pathology of opioids and their pharmacokinetics. However, it is unclear how opioids modulate the gut microbiome and metabolome. Our study, showing opioid modulation of gut homeostasis in mice, suggests that medical interventions to ameliorate the consequences of drug use/abuse will provide potential therapeutic and diagnostic strategies for opioid-modulated intestinal infections. The study of morphine’s modulation of the gut microbiome and metabolome will shed light on the toxicological pathology of opioids and its role in the susceptibility to infectious diseases.
Sex, the states of being female or male, potentially interacts with all xenobiotic exposures, both inadvertent and deliberate, and influences their toxicokinetics (TK), toxicodynamics, and outcomes. Sex differences occur in behavior, exposure, anatomy, physiology, biochemistry, and genetics, accounting for female–male differences in responses to environmental chemicals, diet, and pharmaceuticals, including adverse drug reactions (ADRs). Often viewed as an annoying confounder, researchers have studied only one sex, adjusted for sex, or ignored it. Occupational epidemiology, the basis for understanding many toxic effects in humans, usually excluded women. Likewise, Food and Drug Administration rules excluded women of childbearing age from drug studies for many years. Aside from sex-specific organs, sex differences and sex x age interactions occur for a wide range of disease states as well as hormone-influenced conditions and drug distribution. Women have more ADRs than men; the classic sex hormone paradigm (gonadectomy and replacement) reveals significant interaction of sex and TK including absorption, distribution, metabolisms, and elimination. Studies should be designed to detect sex differences, describe the mechanisms, and interpret these in a broad social, clinical, and evolutionary context with phenomena that do not differ. Sex matters, but how much of a difference is needed to matter remains challenging.
In inhalation toxicity studies, drug-induced lesions are frequently reported in the larynx and sometimes at the tracheal bifurcation (carina) in the rat, but less so in the dog or monkey, bringing into question the relevance of these rodent findings for humans. The rat larynx is widely considered to be more sensitive than that of the dog and monkey in its response to inhaled xenobiotics, although we could find no published data to support this. In this review, data from 52 inhalation studies involving rodent and nonrodent species were collated and reviewed. These data showed that the rodent larynx, and to a lesser extent the carina, was far more commonly affected by treatment than those of the nonrodent. This review indicates the greater susceptibility of the rodent larynx and carina and emphasizes their lack of relevance for man. Observations and data suggest that the human larynx is much closer to the beagle dog and cynomolgus monkey in its response to inhaled xenobiotics and that greater clinical relevance should be placed on any specific findings in these animal models.
Drug-induced hypersensitivity reactions can significantly impact drug development and use. Studies to understand risk factors for drug-induced hypersensitivity reactions have identified genetic association with specific human leukocyte antigen (HLA) alleles. Interestingly, drug-induced hypersensitivity reactions can occur in nonhuman primates; however, association between drug-induced hypersensitivity reactions and major histocompatibility complex (MHC) alleles has not been described. In this study, tissue samples were collected from 62 cynomolgus monkeys from preclinical studies in which 9 animals had evidence of drug-induced hypersensitivity reactions. Microsatellite analysis was used to determine MHC haplotypes for each animal. A total of 7 haplotypes and recombinant MHC haplotypes were observed, with distribution frequency comparable to known MHC I allele frequency in cynomolgus monkeys. Genetic association analysis identified alleles from the M3 haplotype of the MHC I B region (B*011:01, B*075:01, B*079:01, B*070:02, B*098:05, and B*165:01) to be significantly associated (2 test for trend, p < 0.05) with occurrence of drug-induced hypersensitivity reactions. Sequence similarity from alignment of alleles in the M3 haplotype B region and HLA alleles associated with drug-induced hypersensitivity reactions in humans was 86% to 93%. These data demonstrate that MHC alleles in cynomolgus monkeys are associated with drug-induced hypersensitivity reactions, similar to HLA alleles in humans.
Cytological bone marrow evaluation is utilized in nonclinical toxicology studies to characterize hematopoietic effects when the combined interpretation of histologic and complete blood count data does not yield sufficient information. Results from cytological bone marrow examination should be interpreted in the context of variability observed in concurrent control animals with consideration of cytologist experience and historical/published data. Cytological bone marrow differential counts and cellular morphologic findings from 130 (66 male, 64 female) healthy control cynomolgus monkeys from nonclinical toxicology studies were retrospectively analyzed. Myeloid to erythroid (M:E) ratios and the percentage of total cells for each cell type were determined from differential cell count data. M:E ratios ranged from 0.6:1 to 2.3:1. Percentages of total granulocytic cells, total erythroid cells, and lymphocytes ranged from 26.6% to 60.6%, 25.7% to 52.2%, and 5.5% to 40.4%, respectively. Monocytes, plasma cells, mast cells, and mitotic figures were typically <1% of total cells. Notable morphologic findings included occasional giant neutrophilic metamyelocytes and band neutrophils, ring-shaped band neutrophil nuclei, metarubricyte nuclear blebbing and binucleation, multiple or nonfused megakaryocyte nuclei, and emperipolesis. These results represent cytological bone marrow findings from healthy control cynomolgus monkeys utilized in nonclinical toxicology studies and provide insight into expected background variability.
The continuing education course on integrating clinical and anatomical pathology data was designed to communicate the importance of using a weight of evidence approach to interpret safety findings in toxicology studies. This approach is necessary, as neither clinical nor anatomic pathology data can be relied upon in isolation to fully understand the relationship between study findings and the test article. Basic principles for correlating anatomic pathology and clinical pathology findings and for integrating these with other study end points were reviewed. To highlight these relationships, a series of case examples, presented jointly by a clinical pathologist and an anatomic pathologist, were used to illustrate the collaborative effort required between clinical and anatomical pathologists. In addition, the diagnostic utility of traditional liver biomarkers was discussed using results from a meta-analysis of rat hepatobiliary marker and histopathology data. This discussion also included examples of traditional and novel liver and renal biomarker data implementation in nonclinical toxicology studies to illustrate the relationship between discrete changes in biochemistry and tissue morphology.
Age, and in particular young age, can significantly impact the response to toxicants in animals and can greatly influence the interpretation of tissue changes by the toxicologic pathologist. Although this applies to multiple organ systems, the current review focuses on the male reproductive system. When performing microscopic evaluation of male reproductive organs, the toxicologic pathologist must be aware of the dynamic changes in histomorphology, predominantly driven by timed hormonal alterations, at various life stages. Specific challenges pathologists face are understanding the appearance of male reproductive tissues throughout the neonatal, infantile, and juvenile developmental periods, recognizing when normal looks abnormal during tissue development, defining sexual maturity, and working with high interanimal variability in maturation rate and histologic appearance in developing large laboratory animals, such as nonhuman primates, dogs, and pigs. This review describes postnatal development of the male reproductive system in the rat, demonstrates how assessing toxicity during a defined window of postnatal development in the rat may improve definition of toxicant timing and targets, and discusses challenges associated with the interpretation of toxicity in immature large animal species. The emphasis is on key age-related characteristics that influence the interpretation of tissue changes by the toxicologic pathologist.
The immune system is a critical component in defense against viral, bacterial, parasitic, and fungal diseases. Immunological mechanisms, including immunological mediators, innate immunity, cell-mediated immunity, and humoral-mediated immunity, serve to maintain homeostasis and protect the host from disease. Immunological variation can impact defense mechanisms, however. Two factors in particular that can influence immune function are the single nucleotide polymorphisms (SNPs) and aging. SNPs affecting inflammatory cytokines are an important modifier involved in a number of diseases such as asthma, periodontal disease, atherosclerosis, diabetic retinopathy, psoriasis, and osteoporosis. Age-related alterations to the immune system have also been studied and documented. The genetic makeup of different strains of mice and the age of these different strains cause large differences in susceptibility to infection, with influenza virus infection among the most widely studied. The mechanism of these differences due to either genetics or age is not known but can be investigated in strain- and age-specific infectious disease models.
The neurotransmitter glutamate causes excitotoxicity in the human retina. In neonatal rats, the degree of glutamate-induced retinal damage depends on age at administration. To elucidate the sensitivity to glutamate on various developing stage of retina, we investigated glutamate-induced retinal damage and glutamate target cells on each postnatal day (PND). Newborn rats received a single subcutaneous administration of
Ozone is an irritating gas found in photochemical smog. Epidemiological associations have been made between the onset of asthma and childhood exposures to increasing levels of ambient ozone (i.e., air pollutant–induced nonatopic asthma). Individuals, however, vary in their susceptibility to this outdoor air pollutant, which may be due, in part, to their genetic makeup. The present study was designed to test the hypothesis that there are murine strain-dependent differences in pulmonary and nasal pathologic responses to repeated ozone exposures. C57BL/6NTac and BALB/cNTac mice were exposed to 0 or 0.8 ppm ozone, 4 hr/day, for 9 consecutive weekdays. In both strains of mice, ozone induced eosinophilic inflammation and mucous cell metaplasia in the nasal and pulmonary airways. Lungs of ozone-exposed C57BL/6NTac mice, however, had greater eosinophilic inflammation, mucous cell metaplasia, and expression of genes related to type 2 immunity and airway mucus hypersecretion, as compared to similarly exposed BALB/cNTac mice. Ozone-exposed C57BL/6NTac mice also had greater eosinophilic rhinitis but a similar degree of mucous cell metaplasia in nasal epithelium, as ozone-exposed BALB/cNTac mice. These findings suggest that nonatopic individuals may differ in their inflammatory and epithelial responses to repeated ozone exposures that are due, in part, to genetic factors.
Veterinary clinical pathologists are well positioned via education and training to assist in investigations of unexpected results or increased variation in clinical pathology data. Errors in testing and unexpected variability in clinical pathology data are sometimes referred to as "laboratory errors." These alterations may occur in the preanalytical, analytical, or postanalytical phases of studies. Most of the errors or variability in clinical pathology data occur in the preanalytical or postanalytical phases. True analytical errors occur within the laboratory and are usually the result of operator or instrument error. Analytical errors are often ≤10% of all errors in diagnostic testing, and the frequency of these types of errors has decreased in the last decade. Analytical errors and increased data variability may result from instrument malfunctions, inability to follow proper procedures, undetected failures in quality control, sample misidentification, and/or test interference. This article (1) illustrates several different types of analytical errors and situations within laboratories that may result in increased variability in data, (2) provides recommendations regarding prevention of testing errors and techniques to control variation, and (3) provides a list of references that describe and advise how to deal with increased data variability.
Naphthoquine phosphate (NP) was considered as a partner drug with a promising antimalarial drug candidate. Here we report unexpected adverse clinical signs and microscopic findings in a canine pilot toxicology study with NP. Male and female dogs were dosed daily by oral gavage with NP at 2, 10, or 50 mg/kg/day for a maximum of 14 days. NP was not tolerated at ≥10 mg/kg/day; several animals were sacrificed in moribund condition and marked neurological clinical signs were noted at 50 mg/kg/day. The main microscopic observation was central nervous system vasculocentric inflammation (mainly lymphocytes and macrophages) in the white and gray matter of various regions of the brain at ≥2 mg/kg/day and at lower incidence in the spinal cord at ≥10 mg/kg/day. Vasculocentric microscopic changes predominantly centered on the centrilobular vein were also observed in the liver at ≥2 mg/kg/day. Females were more sensitive than males with comparable NP plasma exposure. In conclusion, under the conditions of this study, the administration of NP to dogs via daily oral gavage for up to 2 weeks was not tolerated causing moribundity, marked neurological clinical signs, and vasculocentric microscopic changes in the central nervous system and the liver.
A number of factors related to study design have the potential to impact clinical pathology test results during the conduct of nonclinical safety studies. A thorough understanding of these factors is paramount in drawing accurate conclusions from clinical pathology data generated during such studies, particularly when attempting to make the distinction between test article and nontest article–related effects. Study design and conduct variables with potential to impact clinical pathology data discussed in this overview include those related to species and test system, animal age, animal care and husbandry practices, fasting, acclimatization periods, effects of transportation and stressors, route of administration, effects of in-life and surgical procedures, influence of study length, timing of blood collections, impact of vehicle/formulation composition, and some general concepts related to drug class. The material presented here is a summary based on information presented at the 35th Annual Symposium of the Society of Toxicologic Pathology (June 2016), during Symposium Session 2 titled "Deciphering Sources of Variability in Clinical Pathology—It’s Not Just about the Numbers."
The 2016 annual National Toxicology Program Satellite Symposium, entitled "Pathology Potpourri" was held in San Diego, CA, at the Society of Toxicologic Pathology’s (STP) 35th annual meeting. The goal of this symposium was to present and discuss challenging diagnostic pathology and/or nomenclature issues. This article presents summaries of the speakers’ talks, along with select images that were used by the audience for voting and discussion. Some lesions and topics covered during the symposium included malignant glioma and histiocytic sarcoma in the rodent brain; a new statistical method designed for histopathology data evaluation; uterine stromal/glandular polyp in a rat; malignant plasma cell tumor in a mouse brain; Schwann cell proliferative lesions in rat hearts; axillary schwannoma in a cat; necrosis and granulomatous inflammation in a rat brain; adenoma/carcinoma in a rat adrenal gland; hepatocyte maturation defect and liver/spleen hematopoietic defects in an embryonic mouse; distinguishing malignant glioma, malignant mixed glioma, and malignant oligodendroglioma in the rat; comparison of mammary gland whole mounts and histopathology from mice; and discussion of the International Harmonization of Nomenclature and Diagnostic Criteria collaborations.
The title of the 2016 Society of Toxicologic Pathology (STP) Symposium was the "Basis and Relevance of Variation in Toxicologic Responses." Many factors may contribute to variation in toxicologic responses and can confound results, complicate interpretation of data, interfere with reproducibility, and make extrapolation to humans problematic. This brief overview summarizes speaker presentations from each session which describes important factors that may impact the interpretation of nonclinical discovery and developmental toxicity studies. In addition, summaries of the Continuing Education (CE) courses and other educational events that occurred during the Symposium are highlighted.
Mice and humans branched from a common ancestor approximately 80 million years ago. Despite this, mice are routinely utilized as animal models of human disease and in drug development because they are inexpensive, easy to handle, and relatively straightforward to genetically manipulate. While this has led to breakthroughs in the understanding of genotype–phenotype relationships and in the identification of therapeutic targets, translation of beneficial responses to therapeutics from mice to humans has not always been successful. In a large part, these differences may be attributed to variations in the alignment of protein expression and signaling in the immune systems between mice and humans. Well-established inbred strains of "The Laboratory Mouse" vary in their immune response patterns as a result of genetic mutations and polymorphisms arising from intentional selection for research relevant traits, and even closely related substrains vary in their immune response patterns as a result of genetic mutations and polymorphisms arising from genetic drift. This article reviews some of the differences between the mouse and human immune system and between inbred mouse strains and shares examples of how these differences can impact the usefulness of mouse models of disease.
The objectives of this session were to explore causes of variability in clinical pathology data due to preanalytical and analytical variables as well as study design and other procedures that occur in toxicity testing studies. The presenters highlighted challenges associated with such variability in differentiating test article–related effects from the effects of experimental procedures and its impact on overall data interpretation. These presentations focused on preanalytical and analytical variables and study design–related factors and their influence on clinical pathology data, and the importance of various factors that influence data interpretation including statistical analysis and reference intervals. Overall, these presentations touched upon potential effect of many variables on clinical pathology parameters, including animal physiology, sample collection process, specimen handling and analysis, study design, and some discussion points on how to manage those variables to ensure accurate interpretation of clinical pathology data in toxicity studies. This article is a brief synopsis of presentations given in a session entitled "Deciphering Sources of Variability in Clinical Pathology—It’s Not Just about the Numbers" that occurred at the 35th Annual Symposium of the Society of Toxicologic Pathology in San Diego, California.
Drug-induced phospholipidosis is characterized by intracellular accumulation of phospholipids with lamellar bodies in cells exposed to xenobiotics. Demonstration of the lamellar bodies by transmission electron microscopy (TEM) is the hallmark for a definitive diagnosis of phospholipidosis. However, the preparation of tissue samples for TEM and their ultrastructural evaluation are technically challenging and time consuming. Paraphenylenediamine (PPD) is essentially a fat stain, and the staining mechanism is based upon the osmication of unsaturated lipids. Thus, the application of PPD staining to osmicated tissue samples is considered an optimal way to identify lipids. We evaluated the potential of PPD staining to localize phospholipid accumulations on osmium-fixed semi-thin tissue sections of the lung, kidney, and liver, which were affected with phospholipidosis, under a light microscope. PPD staining revealed the presence of PPD positive dark fine granular material in the cytoplasm for all affected tissues examined, which correlated ultrastructurally with lamellar bodies as well as a light microscopic finding of cytoplasmic vacuolation. The great advantage of PPD is that it can be incorporated into the protocol for standard TEM tissue preparation and significantly improve the efficiency of TEM work. In conclusion, PPD provides a simple, sensitive, and reliable method for visualizing phospholipid accumulation on light microscopy and represents an easy tool to study drug-induced phospholipidosis.
Microminipigs are becoming increasingly attractive alternatives for various experimental applications, such as general toxicology studies, owing to their manageable size. However, there are limited studies on the male reproductive organs of microminipigs, particularly on the histological aspects of sexual maturity. To clarify the development of male reproductive organs, 35 male microminipigs, aged 0 to 12 months, were used in this study. Histological and histomorphological evaluation was performed based on spermatogenic development, measurement of tubular structure in testes and epididymides, and histological progress of accessory glands. In addition, spontaneous testicular changes were quantitatively assessed. Histologically, male microminipigs sexually matured around 4.5 months of age, when spermatogenesis in testes and structural development in genital organs were completed. Spontaneous testicular changes occurred in all the animals investigated. Multinucleated giant cell was most commonly observed, followed by hypospermatogenesis and tubular atrophy/hypoplasia. However, the number of affected tubules was less than 1% in testes after 4.5 months of age, suggesting that the influence of these changes on evaluation of toxicity studies may be minimal. It is preferable to use sexually mature animals in toxicology studies; therefore, the information obtained by the present study will be helpful for future toxicity evaluations in microminipigs.
One of the principal challenges facing a toxicologic pathologist is to determine and differentiate a true adverse effect from a nonadverse or an adaptive response. Recent publications from the Society of Toxicologic Pathology (STP) and the European STP provide guidance for determining and communicating adversity in nonclinical toxicology studies. In order to provide a forum to inform and engage in a discussion on this important topic, a continuing education (CE) course was held during the 2016 STP Annual meeting in San Diego, CA. The lectures at this course provided guidance on determining and communicating adversity using case studies involving both clinical pathology and anatomic pathology. In addition, one talk also focused on data quality, study design, and interpretation of artifacts that could hinder the determination of adversity. The CE course ended with a talk on understanding adversity in preclinical studies and engaging the regulatory agencies in the decision-making process. This manuscript is designed to provide brief summaries of all the talks in this well-received CE course.
The microbiome is increasingly recognized as a critical component in human development, health, and disease. Its relevance to toxicology and pharmacology involves challenges to current concepts related to absorption, metabolism, gene:environment, and pathways of response. Framing testable hypotheses for experimental and epidemiological studies will require attention to study designs, biosampling, data analysis, and attention to confounders.
This article describes acute toxicity data in cynomolgus monkeys following oral treatment with vildagliptin, a dipeptidyl peptidase-4 inhibitor. Acute toxicity symptoms in cynomolgus monkeys include edema formation of the extremities, tails, and face associated with skeletal muscle necrosis, and elevations of lactate dehydrogenase, creatine kinase, alanine transaminase, and aspartate aminotransferase activities in the serum; hypothermia; hypotension; tachycardia; moribundity; and death in a few isolated instances. In surviving animals, symptoms were reversible even if treatment was continued. Cynomolgus monkeys from Mauritius appear more sensitive than monkeys of Asian origin. The underlying mechanism(s) of these symptoms in cynomolgus monkeys is currently not well understood, although a vascular mechanism including initial vasoconstriction and subsequent vascular leakage in distal extremities may play a role. The monkey data are reviewed and discussed in the context of other preclinical and clinical data, and it is concluded that acute toxicity following vildagliptin treatment is a monkey-specific phenomenon without relevance for humans.
Toxicologic pathologists must evaluate tissues of immature animals from a number of types of nonclinical toxicity studies. The pathologist who is familiar with normal postnatal organ development is in a better position to appropriately detect and differentiate between abnormal, delayed, or precocious development. Vacuolation and apoptosis in multiple tissue types are normal components of development that could influence the interpretation of some tissues. Unique postnatal features such as the germal matrix in the brain, gonocytes in the testes, and saccules in the lung may complicate the histopathological evaluation. With the knowledge of normal organ development and critical windows therein, it is possible to design targeted studies to identify xenobiotic toxicity.
It has been long established that not only the species but also the strain and supplier of rodents used in preclinical safety studies can have a significant impact on the outcome of studies due to variability in their genetic background and thus spontaneous pathologic findings. In addition, local husbandry, housing, and other environmental conditions may have effects on the development and expression of comorbidities, particularly in longer-term or chronic studies. More recently, similar effects related to the source, including genetic and environmental variability, have been recognized in cynomolgus macaques (Macaca fascicularis). The increased use of cynomolgus macaques from various sources of captive-bred animals (including nonnative, U.S./European Union-based breeding facilities or colonies) can affect study design and study results and outcome. It is important to acknowledge and understand the impact of this variability on the results and interpretation of research studies. This review includes recent examples where variability of preclinical animal models (rats and monkeys) affected the postmortem observations highlighting its relevance to study design or interpretation in safety studies.
Revision of the International Council for Harmonization (ICH) S1 guidance for rat carcinogenicity studies to be more selective of compounds requiring a 2-year rat carcinogenicity study has been proposed following extensive evaluation of rat carcinogenicity and chronic toxicity studies by industry and drug regulatory authorities. To inform the ICH S1 expert working group in their potential revision of ICH S1, a prospective evaluation study was initiated in 2013, in which sponsors would assess the pharmacologic and toxicologic findings present in the chronic toxicity studies and predict a positive or negative carcinogenicity outcome using a weight of evidence argument (a carcinogenicity assessment document [CAD]). The Scientific and Regulatory Policy Committee was asked by the Society of Toxicology Pathology (STP) executive committee to track these changes with ICH S1 and inform the STP membership of status changes. This commentary is intended to provide a brief summary of recent changes to the CAD guidance and highlight the importance of STP membership participation in the process of CAD submissions.
Evaluation of the female reproductive system in a general toxicity setting can be challenging for the toxicologic pathologist due to the cyclic nature of the estrous and menstrual cycles, timing of puberty and reproductive senescence, and species differences. Age in particular can have a significant impact on the histologic appearance of the female reproductive system and create challenges when trying to distinguish test article–related findings from normal developmental or senescent changes. This review describes the key physiologic and histologic features of immaturity, the transition through puberty, sexual maturity, and reproductive senescence in the female reproductive system, with an emphasis on practical applications for the toxicologic pathologist, and includes recommendations for distinguishing and documenting these developmental periods. Rats and cynomolgus monkeys are used as examples throughout with correlations to clinically observed end points to better aid the toxicologic pathologist in understanding how age may impact study interpretation.
Decades of research, including the 1996 Nobel Prize in Medicine, confirm the evolutionary and immunological importance of CD8 T lymphocytes (TCD8+) that target peptides bound by the highly variable major histocompatibility complex class I (MHC-I) proteins. However, their perceived importance has varied dramatically over the past decade. Regardless, there remains myriad reasons to consider the diversity of MHC-I alleles and the TCD8+ that target them as enormously important in infectious disease research. Thus, understanding these molecules in the best animal models of human disease could be a necessity for optimizing the translational potential of these models. Knowledge of macaque MHC has substantially improved their utility for modeling HIV and could aid in modeling other viruses as well, both in the context of distribution of alleles across treatment groups in vaccine trials and in deciphering mechanisms of immune control of pathogens for which specific MHC alleles demonstrate differential impacts on disease.
Although interpretation and description of clinical pathology test results for any preclinical safety assessment study should employ a consistent standard approach, companies differ regarding that approach and the appearance of the end product. Some rely heavily on statistical analysis, others do not. Some believe reference intervals are important, most do not. Some prefer severity of effects be described by percentage differences from, or multiples of, baseline or control, others prefer only word modifiers. Some expect a definitive decision for every potential effect, others accept uncertainty. This commentary addresses these differences and underscores the need for flexibility in a "consistent standard approach" because the conditions of every study are unique. This article constitutes an overview of material originally presented at Session 2 of the 2016 Society of Toxicologic Pathology Annual Symposium.
Regulatory studies of developmental and reproductive toxicity (DART) studies have remained largely unchanged for decades, with exposures occurring at various phases of the reproductive cycle and toxicity evaluations at different ages/times depending on the study purpose. The National Toxicology Program has conducted studies examining the power to detect adverse effects where there is a prenatal exposure, but evaluations occur postnatally. In these studies, examination is required of only 1 male and female pup from each litter beyond weaning. This provides poor resolving power to detect rare events (e.g., reproductive tract malformations). If an adverse effect is detected, there is little confidence in the shape of the dose–response curve (and the Benchmark Dose or No Observed Adverse Effect Level [NOAEL]). We have developed a new protocol to evaluate DART, the modified one generation study, with exposure commencing with pregnant animals and retention of 4 males and females from each litter beyond weaning to improve statistical power. These animals can be allocated to specific cohorts that examine subchronic toxicity, teratology, littering, and neurobehavioral toxicity in the same study. This approach also results in a reduction in animal numbers used, compared with individual stand-alone studies, and offers increased numbers of end points evaluated compared with recent Organization for Economic Cooperation and Development proposals.
The overarching theme of the 2016 Society of Toxicology Pathology’s Annual Symposium was "The Basis and Relevance of Variation in Toxicologic Responses." Session 4 focused on genetic variation as a potential source for variability in toxicologic responses within nonclinical toxicity studies and further explored how knowledge of genetic traits might enable targeted prospective and retrospective studies in drug development and human health risk assessment. In this session, the influence of both genetic sequence variation and epigenetic modifications on toxicologic responses and their implications for understanding risk were explored. In this overview, the presentations in this session will be summarized, with a goal of exploring the ramifications of genetic and epigenetic variability within and across species for toxicity studies and disseminating information regarding novel tools to harness this variability to advance understanding of toxicologic responses across populations.
A cutaneous response (localized swelling and/or erythema of the skin) has been noted in dog toxicology studies in which multiple, unrelated compounds were administered orally with copovidone as a vehicle. The response has been noted in studies with 6 different test items that are structurally unrelated and span several different therapeutic indications spanning an approximate 6-year period (2009–2015). A factor common among the studies is the formulation—a copovidone amorphous solid dispersion (ASD). Cutaneous responses have not been observed in dogs administered non-ASD formulations of the same test items but have occasionally been noted in placebo (copovidone control) dogs. Polyvinylpyrrolidone (a polymer of one of the primary components of copovidone) has been reported to result in similar findings in dogs when administered by the intravenous route. Considerations for the role of copovidone and the potential role of histamine in the cutaneous changes are outlined.
There is a great need for improved diagnostic and prognostic accuracy of potential cardiac toxicity in drug development. This study reports the evaluation of several commercially available biomarker kits by 3 institutions (SRI, Eli Lilly, and Pfizer) for the discrimination between myocardial degeneration/necrosis and cardiac hypertrophy as well as the assessment of the interlaboratory and interplatform variation in results. Serum concentrations of natriuretic peptides (N-terminal pro-atrial natriuretic peptide [NT-proANP] and N-terminal pro-brain natriuretic peptide [NT-proBNP]), cardiac and skeletal troponins (cTnI, cTnT, and sTnI), myosin light chain 3 (Myl3), and fatty acid binding protein 3 (FABP3) were assessed in rats treated with minoxidil (MNX) and isoproterenol (ISO). MNX caused increased heart-to-body weight ratios and prominent elevations in NT-proANP and NT-proBNP concentrations detected at 24-hr postdose without elevation in troponins, Myl3, or FABP3 and with no abnormal histopathological findings. ISO caused ventricular leukocyte infiltration, myocyte fibrosis, and necrosis with increased concentrations of the natriuretic peptides, cardiac troponins, and Myl3. These results reinforce the advantages of a multimarker strategy in elucidating the underlying cause of cardiac insult and detecting myocardial tissue damage at 24-hr posttreatment. The interlaboratory and interplatform comparison analyses also showed that the data obtained from different laboratories and platforms are highly correlated and reproducible, making these biomarkers widely applicable in preclinical studies.
Permanent vascular catheterization for intravascular access is one of the most commonly applied techniques used on rodents in pharmacology studies. However, use of the intravascular catheters is complicated by nontolerance due to thromboembolic disease and sepsis. We have undertaken an extensive pathologic and clinical analysis of an intravascular catheterization model in Wistar Han and Sprague-Dawley rats, with a particular focus on carotid artery catheterization with or without jugular vein catheterization, in order to define the pathologic mechanisms behind nontolerance and define clinical end points to ensure maximal animal welfare. Further, we have explored various potential solutions to increase the tolerance of the procedure. In these studies, indwelling catheters were found to cause a high degree of thromboembolic disease with infarction in the brain, cecal tip, and kidneys being the primary causes of nontolerance. Loss of greater than 10% body weight was determined to be the most sensitive indicator of nontolerance and was closely correlated with degree of renal parenchymal loss. Sepsis was noted as a very rare complication, indicating that routine aseptic surgical techniques are adequate for preventing this complication.
There are currently no reports describing mammary gland development in the Harlan Sprague-Dawley (HSD) rat, the current strain of choice for National Toxicology Program (NTP) testing. Our goals were to empower the NTP, contract labs, and other researchers in understanding and interpreting chemical effects in this rat strain. To delineate similarities/differences between the female and male mammary gland, data were compiled starting on embryonic day 15.5 through postnatal day 70. Mammary gland whole mounts, histology sections, and immunohistochemically stained tissues for estrogen, progesterone, and androgen receptors were evaluated in both sexes; qualitative and quantitative differences are highlighted using a comprehensive visual timeline. Research on endocrine disrupting chemicals in animal models has highlighted chemically induced mammary gland anomalies that may potentially impact human health. In order to investigate these effects within the HSD strain, 2,3,7,8-tetrachlorodibenzo-p-dioxin, diethylstilbestrol, or vehicle control was gavage dosed on gestation day 15 and 18 to demonstrate delayed, accelerated, and control mammary gland growth in offspring, respectively. We provide illustrations of normal and chemically altered mammary gland development in HSD male and female rats to help inform researchers unfamiliar with the tissue and may facilitate enhanced evaluation of both male and female mammary glands in juvenile toxicity studies.
The potential of chemicals to alter susceptibility to mammary tumor formation is often assessed using a carcinogen-induced study design in various rat strains. The rate of mammary gland (MG) development must be considered so that the timing of carcinogen administration is impactful. In this study, in situ MG development was assessed in females of the Harlan Sprague-Dawley (Hsd:SD), Charles River Sprague-Dawley (Crl:SD), and Charles River Long-Evans (Crl:LE) rat strains at postnatal days 25, 33, and 45. Development was evaluated by physical assessment of growth parameters, developmental scoring, and quantitative morphometric analysis. Although body weight (BW) was consistently lower and day of vaginal opening (VO) occurred latest in female Hsd:SD rats, they exhibited accelerated pre- and peripubertal MG development compared to other strains. Glands of Crl:SD and Crl:LE rats exhibited significantly more terminal end buds (TEBs) and TEB/mm than Hsd:SD rats around the time of VO. These data suggest a considerable difference in the rate of MG development across commonly used strains, which is independent of BW and timing of VO. In mammary tumor induction studies employing these strains, administration of the carcinogen should be timed appropriately, based on strain, to specifically target the peak of TEB occurrence.
Routine husbandry procedures during animal toxicity studies can result in incidental pathological changes. We report on trauma-induced hepatopathy in newborn Göttingen minipigs. Sixty-four neonatal minipigs were allocated to 13- and 26-week treatment arms. There was a 4-week recovery period for both arms. The animals were divided into 2 treatment groups and a vehicle group and were dosed 3 times daily by direct oral administration using a syringe. During the feeding procedure in the first weeks, the animals had to be handled firmly. After 13 weeks, randomly distributed foci of degeneration/necrosis and focal congestion and/or hemorrhage were found in the livers of several animals from all groups. Reduced incidence and severity were evident in the recovery phase, and the lesions were absent after 26 weeks. These changes were considered as related to the manual handling of the animals. Knowledge of these findings is crucial for interpretation of studies utilizing newborn minipigs.
Vascular injury can be induced by different classes of drug candidates, and it can affect the mesenteric vasculature. Sampling of the mesenteric vessels in the rat is crucial for proper assessment of potential adverse or pharmacologic effects of drugs in nonclinical rodent studies. To date, several sampling and processing techniques for the histopathologic evaluation of the mesenteric artery in rodents have been described and used in studies with candidate drugs that may affect the vascular system. However, most of those techniques require a significant amount of time and effort. A less labor-intensive, time-consuming, and expensive technique that allows examination of the mesentery vasculature with abundant longitudinal and cross sections of the vessels when examined microscopically was developed and presented here.
Colitis induced by 2,4,6-Trinitrobenzenesulfonic acid (TNBS) has been used as a model for Crohn’s disease (CD) of inflammatory bowel disease (IBD). Lipocalin-2 (Lcn-2) is an emerging and clinically relevant biomarker of IBD. We investigated the performance of serum and fecal Lcn-2 in the TNBS model of colitis. Female, 7-week-old, BALB/c mice were administered intrarectally phosphate-buffered saline/water or 30% ethanol (vehicle control groups) for 5 days or TNBS for 5 days followed by a 28-day recovery phase. Serum and fecal levels of Lcn-2 were quantified, and effects on body weight, clinical scores, colon weight and length, gross pathology, and histopathology were investigated. Increased serum Lcn-2 levels correlated only with marked to severe inflammation. A clear differentiation in Lcn-2 fecal levels between TNBS-treated and vehicle-treated control mice was most noticeable on days 2 and 3. There was a strong correlation between body weight change, histopathologic scores of inflammation, and/or fecal Lcn-2 levels on days 2 and 5. Both serum and fecal Lcn-2 levels declined over time as the colonic mucosa recovered. Fecal Lcn-2 was found to be a more sensitive biomarker (vs. serum Lcn-2) and was able to discriminate mild, moderate, and severe colonic inflammation.
Inhibition of the mitogen-activated protein kinase/extracellular signal-regulated (MAPK/ERK) pathway is an attractive therapeutic approach for human cancer therapy. In the course of evaluating structurally distinct small molecule inhibitors that target mitogen-activated protein kinase kinase (MEK) and ERK kinases in this pathway, we observed an unusual, dose-related increase in the incidence of green serum in preclinical safety studies in rats. Having ruled out changes in bilirubin metabolism, we demonstrated a 2- to 3-fold increase in serum ceruloplasmin levels, likely accounting for the observed green color. This was not associated with an increase in α-2-macroglobulin, the major acute phase protein in rats, indicating that ceruloplasmin levels increased independently of an inflammatory response. Elevated serum ceruloplasmin was also not correlated with changes in total hepatic copper, adverse clinical signs, or pathology findings indicative of copper toxicity, therefore discounting copper overload as the etiology. Both ERK and MEK inhibitors led to increased ceruloplasmin secretion in rat primary hepatocyte cultures in vitro, and this increase was associated with activation of the Forkhead box, class O1 (FOXO1) transcription factor. In conclusion, increased serum ceruloplasmin induced by MEK and ERK inhibition is due to increased synthesis by hepatocytes from FOXO1 activation and results in the nonadverse development of green serum in rats.
Limited information has been published on the use of cardiac troponin I (cTnI) as a biomarker of cardiac injury in monkeys. The purpose of these studies was to characterize the cTnI response seen in cynomolgus macaques during routine dosing and blood collection procedures typically used in preclinical safety studies and to better understand the pathogenesis of this response. We measured cTnI using two different methods, the Siemens Immulite cTnI assay and the more sensitive Siemens Troponin I-Ultra assay. We were able to demonstrate that after oral, subcutaneous, or intravenous dosing of common vehicles, as well as serial chair restraint for venipuncture blood collection, that minimal to mild transient increases in cTnI could be detected in monkeys with both assays. cTnI values typically peaked at 2, 3, 4, or 6 hr after sham dosing and returned to baseline at 22 or 24 hr. In addition, marked increases in heart rate (HR) and blood pressure (BP) occurred in monkeys during the restraint procedures, which likely initiated the cTnI release in these animals. Monkeys that were very well acclimated to the chairing procedures and had vascular access ports for blood sampling did not have marked increases in HRs and BP or increases in cTnI.
This study was conducted to characterize lung microscopic lesions in control beagle dogs from inhalation and oral gavage toxicity studies, to determine differences associated with the route of administration, and to discuss distinguishing features from compound-induced lung lesions. Samples from 138 control dogs from oral gavage studies and 124 control dogs from inhalation (vehicle control) studies were evaluated microscopically. There was no significant sex-related difference in the incidence of all lesions. Perivascular mononuclear cell infiltration, centriacinar mixed cell infiltration, bronchopneumonia, subpleural septal fibrosis, and alveolar macrophage accumulation were the most common lesions. Aspiration pneumonia was more common in dogs from gavage studies, suggesting reflux after gavage dosing or accidental administration of test formulation as possible causes. Centriacinar mixed cell infiltration was more common in dogs from inhalation studies, suggesting mild irritation by the vehicles used. Vascular lesions, which included pulmonary arteriopathy and smooth muscle mineralization, were observed in a few animals. Some of the spontaneous lesions are similar to lesions induced by test compounds. Compared to spontaneous lesions, compound-induced lesions tend to be multifocal or diffuse, follow a pattern of distribution (e.g., centriacinar, perivascular, and interstitial), show a dose response in the incidence and severity, and may show cell-specific toxicity.
Differences in the responses of conventional and germfree male Sprague-Dawley rats to acute injury induced by alpha-naphthylisothiocyanate (ANIT), a well-characterized biliary epithelial toxicant, were evaluated. Conventional and germfree rats were dosed once orally with 50 mg/kg of ANIT or corn oil alone and serially sacrificed daily for the next 3 days. Germfree rats treated with ANIT tended to have greater increases in virtually all liver and biliary-related analytes compared with conventional rats treated with ANIT; however, significant differences were found only in a few of these analytes including increased bile acids on day 3, total bilirubin on day 4, glutamate dehydrogenase (GLDH) on day 3, and reduced paraoxonase 1 (PON1) on days 2 and 3. Histologic differences between the conventional and germfree rats were modest, but most pronounced on day 2 (24-hr post dosing). Based on subjective scoring, biliary necrosis, neutrophilic cholangitis, and portal tract edema were more severe in germfree rats at 24 hr post dosing compared with conventional rats. Biliary epithelial replication did not differ between treated groups, however. Overall, germfree rats had a modestly greater level of biliary tract injury based on subjective histologic scoring and clinical chemistry measurements following an acute exposure to the well-characterized biliary toxin, ANIT; however, the difference between the ANIT-treated germfree and conventional groups was modest and most evident only within the first day following exposure. These findings suggest that the microbiome did not significantly affect ANIT-induced acute biliary tract injury in the conditions of this study.
Vehicle control Harlan RCCHan™:WIST rats were examined to provide control data for subsequent studies. The rats (180 male and 180 female) were dosed daily via oral gavage with reverse osmosis water for up to 104 weeks. At necropsy, body weights and macroscopic findings were recorded and tissues were collected for histopathology. The mean body weight at terminal sacrifice was 687 g for males and 466 g for females. The overall survival rate was 62% for males and 59% for females. The most common cause of death for males and females found dead or examined following unscheduled euthanasia was pituitary neoplasia with an incidence of 13.9% for males and 18.9% for females. Macroscopic and neoplastic and nonneoplastic microscopic findings are presented by body system.
Identifying environmental exposures that cause adverse mammary gland outcomes in rodents is a first step in disease prevention in humans and domestic pets. "Whole mounts" are an easy and inexpensive tissue preparation method that can elucidate typical or abnormal mammary gland morphology in rodent studies. Here, we propose procedures to facilitate the use of whole mounts for histological identification of grossly noted tissue alterations. We noted lesions in mammary whole mounts from 14-month-old CD-1 mice that were not found in the contralateral gland hematoxylin and eosin (H&E)-stained section. Whole mounts were removed from the slide and carefully processed to produce high-quality histological sections that mirrored the quality of the original H&E-stained section in order to properly diagnose the unidentified gross abnormalities. Incorporation of this method into testing protocols that focus on human relevant chemical and endocrine disruptors exposure will increase the chances of identifying lesions in the gland and reduce the risk of false negative findings. This method can be especially invaluable when lesions are not always palpable during the course of the study or visible at necropsy, or when a single cross section of the mammary gland is otherwise used for detecting lesions.
Sampling blood for toxicokinetic (TK) evaluation in rodents is typically performed using a satellite group of animals to avoid depleting the blood volume and inducing an additional stressor in the main study animals. This practice does not allow for direct comparison of individual animal toxicity to exposure. These studies evaluated serial collection of twelve, 40-µl blood samples from each rat from either a tail clip or a saphenous vein bleed and its impact on toxicologic parameters over 4- and 14-day periods. The results show the feasibility of successfully collecting TK samples from main study animals, using either of the two techniques. Both procedures were amenable to execution by a single technician using dried blood spot sampling. Any changes observed in the primary markers of erythroid mass between the nonbled control rats and repeat sampled rats were minimal and the range of values often overlapped. This technique would improve the quality of data generated from toxicology studies by allowing a direct comparison of systemic exposure to toxicity while at the same time reducing the number of rats by obviating the need for satellite groups.
In July 2015, the U.S. Food and Drug Administration (FDA) posted a new draft guidance entitled "Testicular Toxicity: Evaluation during Drug Development Guidance for Industry," with a 90-day public comment period. As the nonclinical assessment of testicular toxicity often relies on the expert interpretation of pathology affecting the male reproductive tract, this draft guidance is considered directly relevant to the toxicologic pathology community. Therefore, a working group was formed through the Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathologists (STPs) to provide a detailed review of the draft guidance. Specific comments on the guidance were submitted to the FDA by the STP. The draft guidance and all comments received are currently under review with the FDA. This commentary provides a summary of the components of the draft guidance and the comments submitted by the STP with acknowledgment of different perspectives reflected in comments from other respondents.
Historically, pathologists perform manual evaluation of H&E- or immunohistochemically-stained slides, which can be subjective, inconsistent, and, at best, semiquantitative. As the complexity of staining and demand for increased precision of manual evaluation increase, the pathologist’s assessment will include automated analyses (i.e., "digital pathology") to increase the accuracy, efficiency, and speed of diagnosis and hypothesis testing and as an important biomedical research and diagnostic tool. This commentary introduces the many roles for pathologists in designing and conducting high-throughput digital image analysis. Pathology review is central to the entire course of a digital pathology study, including experimental design, sample quality verification, specimen annotation, analytical algorithm development, and report preparation. The pathologist performs these roles by reviewing work undertaken by technicians and scientists with training and expertise in image analysis instruments and software. These roles require regular, face-to-face interactions between team members and the lead pathologist. Traditional pathology training is suitable preparation for entry-level participation on image analysis teams. The future of pathology is very exciting, with the expanding utilization of digital image analysis set to expand pathology roles in research and drug development with increasing and new career opportunities for pathologists.
Spleen tyrosine kinase (Syk) is a nonreceptor tyrosine kinase that is an important signaling enzyme downstream of immunoreceptors containing an intracellular immunoreceptor tyrosine activating motif (ITAM). These receptors encompass a wide variety of biological functions involved in autoimmune disease pathogenesis. There has been considerable interest in the development of inhibitors of the Syk pathway for the treatment of rheumatoid arthritis and systemic lupus erythematosus. We report that Syk inhibition mechanistically caused peri-islet hemorrhages and fibrin deposition in the rat pancreas and that this finding is due to a homeostatic functional defect in platelets. In more limited studies, similar lesions could not be induced in mice, dogs, and cynomolgus monkeys at similar or higher plasma drug concentrations. Irradiation-induced thrombocytopenia caused a phenotypically similar peri-islet pancreas lesion and the formation of this lesion could be prevented by platelet transfusion. In addition, Syk inhibitor-induced lesions were prevented by the coadministration of prednisone. A relatively greater sensitivity of rat platelets to Syk inhibition was supported by functional analyses demonstrating rat-specific differences in response to convulxin, a glycoprotein VI agonist that signals through Syk. These data demonstrate that the Syk pathway is critical in platelet–endothelial cell homeostasis in the peri-islet pancreatic microvasculature in rats.
The microminipig has considerable potential as an animal model to evaluate general toxicity; however, there are few studies on the male reproductive system, particularly regarding spermatogenesis. The objectives of the present study were to clarify the stages of the seminiferous epithelium cycle on the basis of spermiogenesis and to determine the duration of spermatogenesis in the microminipig. Eleven microminipigs from 6 to 9 months of age were used for histological analyses. Spermiogenesis and stages of the seminiferous epithelium cycle were classified according to the degree of acrosomal development as shown by the periodic acid-Schiff reaction. Three of the animals were intravenously injected with 5-bromo-2-deoxyuridine to determine the duration of spermatogenesis by immunohistochemistry. Spermiogenesis was classified into 15 steps according to the morphological development of the acrosome, nucleus, and flagellum. The seminiferous epithelium cycle was classified into 11 stages based on the steps of spermatid development and germ cell associations. The length of the seminiferous epithelium cycle and the overall spermatogenesis process in the microminipig were estimated to be approximately 9.1 and 40.9 days, respectively. The results indicate the potential application of the microminipig in the evaluation of testicular toxicity, such as spermatogenesis disruption, in general toxicity studies.
Quantitative assessment of epidermal nerve fibers (ENFs) has become a widely used clinical tool for the diagnosis of small fiber neuropathies such as diabetic neuropathy and human immunodeficiency virus–associated sensory neuropathy (HIV-SN). To model and investigate the pathogenesis of HIV-SN using simian immunodeficiency virus (SIV)-infected Asian macaques, we adapted the skin biopsy and immunostaining techniques currently employed in human patients and then developed two unbiased image analysis techniques for quantifying ENF in macaque footpad skin. This report provides detailed descriptions of these tools and techniques for ENF assessment in macaques and outlines important experimental considerations that we have identified in the course of our long-term studies. Although initially developed for studies of HIV-SN in the SIV-infected macaque model, these methods could be readily translated to a range of studies involving peripheral nerve degeneration and neurotoxicity in nonhuman primates as well as preclinical investigations of agents aimed at neuroprotection and regeneration.
Standard components of nonclinical toxicity testing for novel pharmaceuticals include clinical and anatomic pathology, as well as separate evaluation of effects on reproduction and development to inform clinical development and labeling. General study designs in regulatory guidances do not specifically mandate use of pathology or reproductive end points across all study types; thus, inclusion and use of these end points are variable. The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology (STP) formed a Working Group to assess the current guidelines and practices on the use of reproductive, anatomic pathology, and clinical pathology end points in general, reproductive, and developmental toxicology studies. The Working Group constructed a survey sent to pathologists and reproductive toxicologists, and responses from participating organizations were collected through the STP for evaluation by the Working Group. The regulatory context, relevant survey results, and collective experience of the Working Group are discussed and provide the basis of each assessment by study type. Overall, the current practice of including specific end points on a case-by-case basis is considered appropriate. Points to consider are summarized for inclusion of reproductive end points in general toxicity studies and for the informed use of pathology end points in reproductive and developmental toxicity studies.
Retinal degeneration due to chronic ambient light exposure is a common spontaneous age-related finding in albino rats, but it can also be related to exposures associated with environmental chemicals and drugs. Typically, light-induced retinal degeneration has a central/hemispherical localization whereas chemical-induced retinal degeneration has a diffuse localization. This study was conducted to identify and characterize treatment-related retinal degeneration in National Toxicology Program rodent bioassays. A total of 3 chronic bioassays in F344/N rats (but not in B6C3F1/N mice) were identified that had treatment-related increases in retinal degeneration (kava kava extract, acrylamide, and leucomalachite green). A retrospective light microscopic evaluation of the retinas from rats in these 3 studies showed a dose-related increase in the frequencies of retinal degeneration, beginning with the loss of photoreceptor cells, followed by the inner nuclear layer cells. These dose-related increased frequencies of degenerative retinal lesions localized within the central/hemispherical region are suggestive of exacerbation of light-induced retinal degeneration.
Terminal body weights (TBWs), thymus weight parameters, and thymus morphology were retrospectively evaluated in 453 cynomolgus monkeys assigned to control groups on nonclinical toxicity studies. Morphology of bone, ovary, and testis/epididymis were used to determine maturity status of individual animals. There was no correlation between TBW and thymus weight (absolute and/or relative to TBW or brain weight). Thymus weight parameters and grades of decreased lymphocytes in the thymus were highly variable in immature animals compared to mature animals. There was also high (up to 11-fold) variability of thymus weight parameters within a given control group on the same study (generally 3 or 4 animals per sex). Several parameters evaluated had more pronounced age-related changes in males when compared to females. Our results demonstrate the inherent variability of thymus weight parameters and morphologic observations for cynomolgus monkeys on toxicology studies. Changes in thymus parameters in cynomolgus monkeys are unreliable indicators of immunomodulation or immunotoxicity in the absence of other relevant findings. Therefore, the thymus parameters commonly evaluated in preclinical safety assessments should not be the primary data set used to determine the presence of a direct test article–related effect on the immune system.
A critical event in embryo development is the proper formation of the vascular system, of which the hepatobiliary system plays a pivotal role. This has led researchers to use transgenic mice to identify the critical steps involved in developmental disorders associated with the hepatobiliary vascular system. Vascular development is dependent upon normal vasculogenesis, angiogenesis, and the transformation of vessels into their adult counterparts. Any alteration in vascular development has the potential to cause deformities or embryonic death. Numerous publications describe specific stages of vascular development relating to various organs, but a single resource detailing the stage-by-stage development of the vasculature pertaining to the hepatobiliary system has not been available. This comprehensive histology atlas provides hematoxylin & eosin and immunohistochemical-stained sections of the developing mouse blood and lymphatic vasculature with emphasis on the hepatobiliary system between embryonic days (E) 11.5–18.5 and the early postnatal period. Additionally, this atlas includes a 3-dimensional video representation of the E18.5 mouse venous vasculature. One of the most noteworthy findings of this atlas is the identification of the portal sinus within the mouse, which has been erroneously misinterpreted as the ductus venosus in previous publications. Although the primary purpose of this atlas is to identify normal hepatobiliary vascular development, potential embryonic abnormalities are also described.
Diethylnitrosamine (DEN) is a chemical broadly used in animal models as a hepatocarcinogen, reported to also cause pulmonary neoplasms in mice. The original objective was to evaluate the impact of a Western diet with or without 10% broccoli on DEN-induced on liver cancer. We administered DEN (45 mg/kg) intraperitoneally to young adult male B6C3F1 mice by 6 weekly injections and evaluated liver cancer 6 months after the DEN treatments. Here, we report unexpected primary tumorigenesis in nasal epithelium, independent of dietary treatment. More than 50% of DEN-treated B6C3F1 mice developed nasal neoplasm-related lesions, not reported previously in the literature. Only one of these neoplasms was visible externally prior to postmortem examination. Intraperitoneal DEN treatment used as a model for liver cancer can have a carcinogenic effect on the nasal epithelium in B6C3F1 mice, which should be carefully monitored in future liver cancer studies.
Gastrointestinal toxicity is dose limiting with many therapeutic and anticancer agents. Real-time, noninvasive detection of markers of toxicity in biofluids is advantageous. Ongoing research has revealed microRNAs as potential diagnostic and predictive biomarkers for the detection of select organ toxicities. To study the potential utility of microRNA biomarkers of intestinal injury in a preclinical toxicology species, we evaluated 3 rodent models of drug-induced intestinal toxicity, each with a distinct mechanism of toxicity. MiR-215 and miR-194 were identified as putative intestinal toxicity biomarkers. Both were evaluated in plasma and feces and compared to plasma citrulline, an established intestinal injury biomarker. Following intestinal toxicant dosing, microRNA changes in feces and plasma were detected noninvasively and correlated with histologic evidence of intestinal injury. Fecal miR-215 and miR-194 levels increased, and plasma miR-215 decreased in a dose- and time-dependent manner. Dose-dependent decreases in plasma miR-215 levels also preceded and correlated positively with plasma citrulline modulation, suggesting miR-215 is a more sensitive biomarker. Moreover, during the drug-free recovery phase, plasma miR-215 returned to predose levels, supporting a corresponding recovery of histologic lesions. Despite limitations, this study provides preliminary evidence that select microRNAs have the potential to act as noninvasive, sensitive, and quantitative biomarkers of intestinal injury.
In order to harmonize diagnostic terminology, confirm diagnostic criteria, and describe aspects of tumor biology characteristic of different tumor types, a total of 165 cases of mesenchyme-related tumors and nephroblastomas of the rat kidney were reexamined from the National Toxicology Program (NTP) Archives. This survey demonstrated that renal mesenchymal tumor (RMT) was the most common spontaneous nonepithelial tumor in the rat kidney, also occurring more frequently in the NTP studies than nephroblastoma. Renal sarcoma was a distinct but very rare tumor entity, representing a malignant, monomorphous population of densely crowded, fibroblast-like cells, in which, unlike RMT, preexisting tubules did not persist. Nephroblastoma was characterized by early death of affected animals, suggesting an embryonal origin for this tumor type. Male and female rats were equally disposed to developing RMT, but most of the cases of nephroblastoma occurred in female rats and liposarcoma occurred mostly in male rats. This survey confirmed discrete histopathological and biological differences between the mesenchyme-related renal tumor types and between RMT and nephroblastoma. Statistical analysis also demonstrated a lack of any relationship of these renal tumor types to test article administration in the NTP data bank.
Epiphyseal growth plate dysplasia (chondrodysplasia) might be considered as the pathognomonic feature of antiangiogenic treatment in preclinical species as it is reliably and dose-responsively induced in rodents and monkeys with vascular endothelial growth factor receptor (VEGFR) inhibitors, fibroblast growth factor (FGF) receptor inhibitors, matrix metalloproteinase inhibitors, and vascular targeting agents. Here we report epiphyseal growth plate dysplasia in juvenile rabbits treated with an oral spleen tyrosine kinase inhibitor induced by off-target antiangiogenic inhibition of VEGF and FGF family kinase receptors. Epiphyseal growth plate dysplasia resulted in weakening and fracturing of the femoral head physis in 6 of 10 male and 1 of 10 female animals as well as microfracturing and dysplasia of the distal femoral articular cartilage in 1 male animal. Fracture lines ran through the zone of hypertrophic cartilage (as well as adjacent zones), were orientated parallel to the physeal plane, and often involved displacement of the femoral head. We would suggest that the high prevalence of growth plate fracture in the rabbit may represent a potential additional adverse risk to those already established for children treated with antiangiogenic therapy.
Conventional chemotherapy treatments for pancreatic cancer are mainly palliative. RNA interference (RNAi)-based drugs present the potential for a new targeted treatment.
The identification of adverse health effects has a central role in the development and risk/safety assessment of chemical entities and pharmaceuticals. There is currently a need for better alignment regarding how nonclinical adversity is determined and characterized. The European Society of Toxicologic Pathology (ESTP) therefore coordinated a workshop to review available definitions of adversity, weigh determining and qualifying factors of adversity based on case examples, and recommend a practical approach to define and characterize adversity in toxicology reports, to serve as a valuable prerequisite for future organ- or lesion-specific workshops planned by the ESTP.
Given the proven utility of natriuretic peptides as serum biomarkers of cardiovascular maladaptation and dysfunction in humans and the high cross-species sequence conservation of atrial natriuretic peptides, natriuretic peptides have the potential to serve as translational biomarkers for the identification of cardiotoxic compounds during multiple phases of drug development. This work evaluated and compared the response of N-terminal proatrial natriuretic peptide (NT-proANP) and N-terminal probrain natriuretic peptide (NT-proBNP) in rats during exercise-induced and drug-induced increases in cardiac mass after chronic swimming or daily oral dosing with a peroxisome proliferator-activated receptor agonist. Male Sprague-Dawley rats aged 8 to 10 weeks were assigned to control, active control, swimming, or drug-induced cardiac hypertrophy groups. While the relative heart weights from both the swimming and drug-induced cardiac hypertrophy groups were increased 15% after 28 days of dosing, the serum NT-proANP and NT-proBNP values were only increased in association with cardiac hypertrophy caused by compound administration. Serum natriuretic peptide concentrations did not change in response to adaptive physiologic cardiac hypertrophy induced by a 28-day swimming protocol. These data support the use of natriuretic peptides as fluid biomarkers for the distinction between physiological and drug-induced cardiac hypertrophy.
N, N-dimethyl-p-toluidine (DMPT; Cas No. 99-97-8), an accelerant for methyl methacrylate monomers in medical devices, is a nasal cavity carcinogen according to a 2-yr cancer study of male and female F344/N rats, with the nasal tumors arising from the transitional cell epithelium. In this study, we exposed male F344/N rats for 5 days to DMPT (0, 1, 6, 20, 60, or 120 mg/kg [oral gavage]) to explore the early changes in the nasal cavity after short-term exposure. Lesions occurred in the nasal cavity including hyperplasia of transitional cell epithelium (60 and 120 mg/kg). Nasal tissue was rapidly removed and preserved for subsequent laser capture microdissection and isolation of the transitional cell epithelium (0 and 120 mg/kg) for transcriptomic studies. DMPT transitional cell epithelium gene transcript patterns were characteristic of an antioxidative damage response (e.g., Akr7a3, Maff, and Mgst3), cell proliferation, and decrease in signals for apoptosis. The transcripts of amino acid transporters were upregulated (e.g., Slc7a11). The DMPT nasal transcript expression pattern was similar to that found in the rat nasal cavity after formaldehyde exposure, with over 1,000 transcripts in common. Molecular changes in the nasal cavity after DMPT exposure suggest that oxidative damage is a mechanism of the DMPT toxic and/or carcinogenic effects.
Occupational exposure to 2,3-butanedione (BD) vapors has been associated with severe respiratory disease leading to the use of potentially toxic substitutes. We compared the reactivity and respiratory toxicity of BD with that of two structurally related substitutes, 2,3-pentanedione (PD) and 2,3-hexanedione (HD). Chemical reactivity of the diketones with an arginine substrate decreased with increasing chain length (BD
Silver nanoparticle (Ag NP) production methods are being developed and refined to produce more uniform Ag NPs through chemical reactions involving silver salt solutions, solvents, and capping agents to control particle formation. These chemical reactants are often present as contaminants and/or coatings on the Ag NPs, which could alter their interactions in vivo. To determine pulmonary effects of citrate-coated Ag NPs, Sprague-Dawley rats were exposed once nose-only to aerosolized Ag NPs (20 nm [C20] or 110 nm [C110] Ag NPs) for 6 hr. Bronchoalveolar lavage fluid (BALF) and lung tissues were obtained at 1, 7, 21, and 56 days postexposure for analyses. Inhalation of Ag NPs, versus citrate buffer control, produced significant inflammatory and cytotoxic responses that were measured in BALF cells and supernatant. At day 7, total cells, protein, and lactate dehydrogenase were significantly elevated in BALF, and peak histopathology was noted after C20 or C110 exposure versus control. At day 21, BALF polymorphonuclear cells and tissue inflammation were significantly greater after C20 versus C110 exposure. By day 56, inflammation was resolved in Ag NP–exposed animals. Overall, results suggest delayed, short-lived inflammatory and cytotoxic effects following C20 or C110 inhalation and potential for greater responses following C20 exposure.
Due to potential misdiagnosis of hyaline glomerulopathy (HG) for amyloidosis, a retrospective study of B6C3F1 mice from the National Toxicology Program (NTP) archives was undertaken to determine whether HG had occurred in prior NTP studies and, if so, whether these 2 glomerular lesions could be routinely discriminated. Kidney slides from 7 amyloid-positive control mice, 2 HG-positive control mice, 3 normal or negative control mice, and 41 potential HG mice (with renal-only deposits previously diagnosed as amyloid) were evaluated using hematoxylin and eosin (H&E), periodic acid Schiff (PAS), Congo red (CR), and Masson’s trichrome (MT) stains. Utilizing these techniques, HG was reliably distinguished from amyloidosis. All 41 potential HG mice had glomerular deposits histochemically inconsistent with amyloid; the deposits were PAS positive and CR negative. Four of the 41 mice were selected for transmission electron microscopy of the glomerular deposits; ultrastructurally, the deposits in these animals were consistent with HG and not amyloid. Our findings indicate that HG is a spontaneous lesion in B6C3F1 mice of low occurrence, is commonly misdiagnosed as amyloidosis, and is more likely than amyloid to cause glomerular deposits in mice without evidence of deposits in other tissues. Also, HG can be distinguished from amyloid on H&E evaluation; however, the distinction is improved with use of PAS or CR staining and/or ultraviolet evaluation.
Atlantic killifish (Fundulus heteroclitus) inhabiting the Atlantic Wood Industries region of the Elizabeth River, Virginia, have passed polycyclic aromatic hydrocarbon (PAH) resistance to their offspring as evidenced by early life stage testing of developmental toxicity after exposure to specific PAHs. Our study focused on environmentally relevant PAH mixtures in the form of Elizabeth River sediment extract (ERSE). Juvenile (5 month) F1 progeny of pollution-adapted Atlantic Wood (AW) parents and of reference site (King’s Creek [KC]) parents were exposed as embryos to ERSE. Liver alterations, including nonneoplastic lesions and microvesicular vacuolation, were observed in both populations. ERSE-exposed KC fish developed significantly more alterations than unexposed KC fish. Interestingly, unexposed AW killifish developed significantly more alterations than unexposed KC individuals, suggesting that AW juveniles are not fully protected from liver disease; rapid growth of juvenile fish may also be an accelerating factor for tumorigenesis. Because recent reports show hepatic tumor formation in adult AW fish, the differing responses from the 2 populations provided a way to determine whether embryo toxicity protection extends to juveniles. Future investigations will analyze older life stages of killifish to determine differences in responses related to chronic disease.
Cumene hydroperoxide (CHP) is a high production volume chemical that is used to generate phenol and acetone. Dermal exposure to CHP was hypothesized to result in systemic tissue toxicity, production of free radicals, and consequent decrease in plasma antioxidant levels. To evaluate the hypothesis and characterize the toxicity of CHP, male and female B6C3F1/N mice and F344/N rats were exposed to varying doses of CHP applied topically for 14 or 90 days. No significant changes in survival or body weight of mice and rats were observed following 14 days of exposure. However, 90 days of CHP exposure at the high dose (12 mg/kg) triggered a significant decrease (–15%) in the body weight of the male rat group only. Irritation of the skin was observed at the site of application and was characterized by inflammation and epidermal hyperplasia. In treated animals, histology of liver tissue, free radical generation, and antioxidant levels in blood plasma were not significantly changed as compared to the corresponding controls. Consistent with the lack of systemic damage, no increase in micronucleated erythrocytes was seen in peripheral blood. In conclusion, topical CHP application caused skin damage only at the application site and did not cause systemic tissue impairment.
Nonacog beta pegol is a 40-kDa polyethylene glycosylated (PEGylated) human recombinant coagulation factor IX, intended for the treatment of hemophilia B. Human coagulation factors are immunogenic in animals; therefore, to evaluate the long-term toxicity of nonacog beta pegol, an immune-deficient, athymic rat (Rowett nude; Crl:NIH-Foxn1rnu ) was used. Rats (n = 216) were given intravenous nonacog beta pegol 0, 40, 150, 600, or 1,200 IU/kg every 5th day for 26 weeks. To avoid infections, the animals were housed in a full-barrier environment with sterilized food and bedding. Standard toxicity end points were unaffected by treatment. All treated animals were exposed to nonacog beta pegol throughout the study, and no animals developed antidrug antibodies. Immunohistochemical staining revealed PEG in choroid plexus epithelial cells in a dose-dependent manner. Transmission electron microscopy showed that PEG was distributed in cytoplasmic vesicles of these cells, with no apparent effect on cellular organelle structures. Fourteen (6.5%) animals were euthanized or died prematurely due to nontreatment-related infections in the urogenital system and skin. In conclusion, the athymic rat is a suitable model for testing chronic toxicity of human proteins that are immunogenic in animals. Nonacog beta pegol was generally well tolerated, with no adverse effect of PEG on choroid plexus epithelial cells.
Modulation of the cell cycle may underlie the toxicologic or pharmacologic responses of a potential therapeutic agent and contributes to decisions on its preclinical and clinical safety and efficacy. The descriptive and quantitative assessment of normal, aberrant, and degenerate mitotic figures in tissue sections is an important end point characterizing the effect of xenobiotics on the cell cycle. Historically, pathologists used manual counting and special staining visualization techniques such as immunohistochemistry for quantification of normal, aberrant, and degenerate mitotic figures. We designed an automated image analysis algorithm for measuring these mitotic figures in hematoxylin and eosin (H&E)-stained sections. Algorithm validation methods used data generated from a subcutaneous human transitional cell carcinoma xenograft model in nude rats treated with the cell cycle inhibitor Eg5. In these studies, we scanned and digitized H&E-stained xenografts and applied a complex ruleset of sequential mathematical filters and shape discriminators for classification of cell populations demonstrating normal, aberrant, or degenerate mitotic figures. The resultant classification system enabled the representations of three identifiable degrees of morphological change associated with tumor differentiation and compound effects. The numbers of mitotic figure variants and mitotic indices data generated corresponded to a manual assessment by a pathologist and supported automated algorithm verification and application for both efficacy and toxicity studies.
The evaluation of inhalation studies in monkeys is often hampered by the scarcity of published information on the relevant nasal anatomy and pathology. We examined nasal cavities of 114 control cynomolgus monkeys from 11 inhalation studies evaluated 2008 to 2013, in order to characterize and document the anatomic features and spontaneous pathology. Compared to other laboratory animals, the cynomolgus monkey has a relatively simple nose with 2 unbranched, dorsoventrally stacked turbinates, large maxillary sinuses, and a nasal septum that continues into the nasopharynx. The vomeronasal organ is absent, but nasopalatine ducts are present. Microscopically, the nasal epithelium is thicker than that in rodents, and the respiratory (RE) and transitional epithelium (TE) rest on a thick basal lamina. Generally, squamous epithelia and TE line the vestibule, RE, the main chamber and nasopharynx, olfactory epithelium, a small caudodorsal region, while TE is observed intermittently along the passages. Relatively high incidences of spontaneous pathology findings, some resembling induced lesions, were observed and included inflammation, luminal exudate, scabs, squamous and respiratory metaplasia or hyperplasia, mucous cell hyperplasia/metaplasia, and olfactory degeneration. Regions of epithelial transition were the most affected. This information is considered helpful in the histopathology evaluation and interpretation of inhalation studies in monkeys.
Prosthetic annuloplasty rings are a common treatment modality for mitral regurgitation, and recently, percutaneous implantation techniques have gained popularity due to their favorable safety profile. Although in common use, biocompatibility of annuloplasty rings has been reported only sparsely in the literature, and none of these reports used the percutaneous technique of implantation. We report on the biocompatibility and the systemic safety of a novel transcatheter mitral valve annuloplasty ring (AMEND™) in 6 minipigs. This device is composed of a nitinol tube surrounded by a braided polyethylene terephthalate fabric tube. The device produced no adverse inflammatory response, showing gradual integration between the metal ring and the fabric by normal host fibrocellular response, leading to complete neoendocardium coverage. There was no evidence for adverse reactions, rejection, or intolerance in the valvular structure. In 2 animals, hemopericardium resulted from the implantation procedure, leading to right-sided cardiac insufficiency with pulmonary edema and liver congestion. The findings reported herein can serve as a case study for the expected healing pathology reactions after implantation of transcatheter mitral valve annuloplasty rings.
We report renal tubular adenomas and a carcinoma in 26-week Tg.rasH2 mouse carcinogenicity studies, which have not been reported to date either at our facility or in other published data. However, during the year 2014, renal tubular tumors were present in 4 studies conducted at our facility. Because of their morphological similarity to the amphophilic–vacuolar (AV) phenotypic variant of renal tubule tumors noted in Sprague-Dawley and Fischer 344 rats, which are thought to be familial, as well as the genetic homogeneity of Tg.rasH2 mice, we tracked the parents of these mice with tumors in each study. The origin of these tumors could not be traced back to any of the parents or even an animal barrier, and these tumors were not attributed to the vehicle or test article. Although the exact mechanism of tumorigenesis was not known, based on the available information, the development of renal tumors in these mice was considered random and spontaneous.
Perfluorohexanoic acid PFHxA a 6-carbon perfluoroalkyl (C6; CAS # 307-24-4), has been proposed as a replacement for the commonly used 8-carbon perfluoroalkyls: perfluorooctanoic acid and perfluorooctanoic sulfonate. PFHxA is not currently a commercial product but rather the ultimate degradation product of C6 fluorotelomer used to make C6 fluorotelomer acrylate polymers. It can be expected that, to a greater or lesser extent, the environmental loading of PFHxA will increase, as C6 fluorotelomer acrylate treatments are used and waste is generated. This article reports on a chronic study (duration 104 weeks) that was performed to evaluate the possible toxicologic and carcinogenic effects of PFHxA in gavage (daily gavage, 7 days per week) treated male and female Sprague-Dawley (SD) rats. In the current study, dosage levels of 0, 2.5, 15, and 100 mg/kg/day of PFHxA (males) and 5, 30, and 200 mg/kg/day of PFHxA (females) were selected based on a previous subchronic investigation. No effects on body weights, food consumption, a functional observational battery, or motor activity were observed after exposure to PFHxA. While no difference in survival rates in males was seen, a dose-dependent decrease in survival in PFHxA-treated female rats was observed. Hematology and serum chemistry were unaffected by PFHxA. PFHxA-related histologic changes were noted in the kidneys of the 200-mg/kg/day group females. Finally, there was no evidence that PFHxA was tumorigenic in male or female SD rats at any of the dosage levels examined.
We have previously indicated that the ideal animal tumor model should mimic the human disease. This means that the investigator should be able to ascertain the influence of host factors on the initiation of tumorigenesis, mimic the susceptibility of tumor response based on age and reproductive history, and determine the response of the tumors induced to chemotherapy. The utilization of experimental models of mammary carcinogenesis in risk assessment requires that the influence of ovarian, pituitary, and placental hormones, among others, as well as overall reproductive events are taken into consideration, since they are important modifiers of the susceptibility of the organ to neoplastic development. Several species, such as rodents, dogs, cats, and monkeys, have been evaluated for these purposes; however, none of them fulfills all the criteria specified previously. Rodents, however, are the most widely used models; therefore, this work will concentrate on discussing the rat rodent model of mammary carcinogenesis.
The National Toxicology Program (NTP) has developed a new flexible study design, termed the modified one generation (MOG) reproduction study. The MOG study will encompass measurements of developmental and reproductive toxicity parameters as well as enable the setting of appropriate dose levels for a cancer bioassay through evaluation of target organ toxicity that is based on test article exposure that starts during gestation. This study design is compared and contrasted with the new Organization for Economic Co-operation and Development (OECD) 443 test guideline, the extended one generation reproduction study. The MOG study has a number of advantages, with a focus on F 1 animals, the generation of adequately powered, robust data sets that include both pre and postnatal developmental toxicity information, and the measurement of effects on reproductive structure and function in the same animals. This new study design does not employ the use of internal triggers in the design structure for the use of animals already on test and is also consistent with the principles of the 3R’s.
Soft tissue filler products have become very popular in recent years, with ever-increasing medical and aesthetic indications. While generally considered safe, the number of reported complications with tissue fillers is growing. Nevertheless, there is no specific animal model that is considered as the gold standard for assessing safety or efficacy of tissue fillers, and there are very little data on interspecies differences in reaction to these products. Here, we report on interspecies differences in reaction to a subcutaneous injectable co-polyester, composed of castor oil and citric acid. Comparison of the histopathological local tissue changes following 1-month postimplantation, indicated that in rats the reaction consisted of cavities, surrounded by relatively thin fibrotic enveloping capsule. In contrast, an unexpected severe inflammatory granulomatous reaction was noticed in Sinclair minipigs. To our knowledge, this is the first report on significant interspecies differences in sensitivity to tissue fillers. It emphasizes the importance of using the appropriate animal model for performing preclinical biocompatibility assays for biodegradable polymers, tissue fillers, and implanted medical devices in general. It also makes the Sinclair minipig subject for scrutiny as an animal model in future biocompatibility studies.
Medulloblastomas (MBs) are thought to be derived from granular cell precursors in the external granular layer (EGL) of the developing cerebellum. Heterozygous patched1 (Ptch1) knockout mice develop MBs that resemble those in humans when the sonic hedgehog (Shh) signaling pathway is activated. The present study was conducted to evaluate postnatal effects of a Shh signaling inhibitor, cyclopamine, on the development of MBs in Ptch1 mice. Ptch1 and wild-type mice were treated daily with subcutaneous cyclopamine at 40 mg/kg or vehicle from postnatal day (PND) 1 to PND14, and the subsequent development of MBs and preneoplastic lesions was examined up to week 12 (W12). Proliferative lesions in the cerebellum, MBs, and preneoplastic lesions were only detected in Ptch1 mice. Cyclopamine treatment resulted in a statistically significant reduction in the incidence and/or area of proliferative lesions at PND14 and 21. The trend of decreasing preneoplastic lesions persisted up to W12. At PND7, cyclopamine treatment reduced the width and proliferation of the EGL regardless of genotype. These results indicate that inhibition of Shh signaling during cerebellar development has prolonged inhibitory potential on MB development in Ptch1 mice. This inhibitory potential might be related to inhibition of EGL proliferation, including preneoplastic MB cells.
Because of the size of the nanoparticles, their detection and exact anatomical localization in tissue samples are very difficult. Consequently, suitable methods are needed to prove their presence, especially co-localized to histological lesions. Therefore, the aim of this study was to investigate whether nanoparticles were detectable in specimens after reprocessing samples from glass slides using the pop-off technique. Tissue slides containing agglomerates of titanium dioxide nanoparticles already visible on a light microscopic level and amorphous silicium dioxide (SiO2) particles not observable in tissue slides were reprocessed. Furthermore, cytospots of bronchoalveolar lavage acquired from rats that previously inhaled carbon nanotubes were used. After reprocessing the samples, they were investigated using transmission electron microscopy. In all the reprocessed samples, the respective nanoparticles were detectable. Even the light microscopically invisible amorphous SiO2 particles were observed as electron dense structures. Titanium and silicium were additionally confirmed in the respective nanoparticles by energy-dispersive X-ray spectroscopy (EDX). In summary, the pop-off technique represents a fast and easy way to detect nanoparticles in histological sections. This enables further characterization of these particles by additional techniques such as EDX, and their direct correlation with light microscopic lesions at exactly the same location is investigated.
Circulating microRNAs (miRNAs) show promise as biomarkers due to their tissue-specific expression and high stability. This study was conducted to investigate whether nervous system–enriched miR-9* and hippocampus-enriched miR-384-5p could be indicators of neurotoxicity in serum. Rats were given a single administration of trimethyltin (TMT) chloride at 6, 9, or 12 mg/kg by gavage, and brain and serum were collected 1, 4, and 7 days after administration. MiR-9* and miR-384-5p levels in serum and hippocampus were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR), and their neurotoxicity detection sensitivities were compared with nervous symptoms, auditory response, and histopathology. TMT caused tremor, hypersensitivity, and decreased auditory response at 12 mg/kg on day 1 and at 9 mg/kg on day 4. Histopathologically, neural cell death and glial reaction were observed in brain (mainly hippocampus) at 12 mg/kg on day 1, 4, and 7 and at 6 and 9 mg/kg on day 4 and 7. MiR-9* and miR-384-5p levels were elevated in serum at 9 and 12 mg/kg on days 4 and 7 (at 9 mg/kg on day 7, miR-9* only) but were not changed in hippocampus. These miRNAs were considered to be elevated with the evolution of neural cell death and were thus considered possible novel indicators of neurotoxicity.
Vaginal exfoliative cytology is commonly used in biomedical and toxicological research to classify the stages of the rodent estrous cycle. However, mouse vaginal exfoliative cytology is commonly used as a stand-alone tool and has not been evaluated in reference to vaginal histology and serum sex hormone levels. In this study, the direct and Giemsa-stained methods of vaginal exfoliative cytology were compared in reference to vaginal fold histology and serum sex hormone levels. Both methods predicted the estrous stages similarly with mean discordance rates of 55%, 77%, 46%, and 31%, for diestrus, proestrus, estrus, and metestrus, respectively. From these results, we conclude that vaginal exfoliative cytology may be used as a general guide to determine the desired estrous stage end point and that a definitive confirmation of the estrous stage should be obtained from evaluation of vaginal fold histology. Confirmation of the stage of the estrous cycle by vaginal fold histology will decrease the variability otherwise introduced by misclassification of estrous cycle stages with vaginal exfoliative cytology.
Better biomarkers are needed to identify, characterize, and/or monitor drug-induced vascular injury (DIVI) in nonclinical species and patients. The Predictive Safety Testing Consortium (PSTC), a precompetitive collaboration of pharmaceutical companies and the U.S. Food and Drug Administration (FDA), formed the Vascular Injury Working Group (VIWG) to develop and qualify translatable biomarkers of DIVI. The VIWG focused its research on acute DIVI because early detection for clinical and nonclinical safety monitoring is desirable. The VIWG developed a strategy based on the premise that biomarkers of DIVI in rat would be translatable to humans due to the morphologic similarity of vascular injury between species regardless of mechanism. The histomorphologic lexicon for DIVI in rat defines degenerative and adaptive findings of the vascular endothelium and smooth muscles, and characterizes inflammatory components. We describe the mechanisms of these changes and their associations with candidate biomarkers for which advanced analytical method validation was completed. Further development is recommended for circulating microRNAs, endothelial microparticles, and imaging techniques. Recommendations for sample collection and processing, analytical methods, and confirmation of target localization using immunohistochemistry and in situ hybridization are described. The methods described are anticipated to aid in the identification and qualification of translational biomarkers for DIVI.
Information about the incidence of spontaneously occurring, nonneoplastic background findings in Syrian hamsters is essential if Syrian hamsters are to be used for toxicity studies. Male and female Syrian hamsters of the strain Han:AURA from the Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM) breeding colony were maintained as control animals for carcinogenicity studies and were examined for the presence of nonneoplastic background findings either when they died or when the study was terminated. The nonneoplastic background lesions observed at an incidence of >50% (high), >25% (moderate), and >10% (low) in either male or female animals or in both sexes in one or more long-term studies are detailed. The results are compared to previous published reports of nonneoplastic, spontaneous background lesions in Syrian hamsters. Background information about the incidence of background lesions in Syrian hamsters on short- and long-term studies is useful to both toxicologists and toxicological pathologists.
Drug-induced vascular Injury (DIVI) is a common preclinical toxicity usually characterized by hemorrhage, vascular endothelial and smooth muscle damage, and inflammation. DIVI findings can cause delays or termination of drug candidates due to low safety margins. The situation is complicated by the absence of sensitive, noninvasive biomarkers for monitoring vascular injury and the uncertain relevance to humans. The Safer And Faster Evidence-based Translation (SAFE-T) consortium is a public–private partnership funded within the European Commission’s Innovative Medicines Initiative (IMI) aiming to accelerate drug development by qualifying biomarkers for drug-induced organ injuries, including DIVI. The group is using patients with vascular diseases that have key histomorphologic features (endothelial damage, smooth muscle damage, and inflammation) in common with those observed in DIVI, and has selected candidate biomarkers associated with these features. Studied populations include healthy volunteers, patients with spontaneous vasculitides and other vascular disorders. Initial results from studies with healthy volunteers and patients with vasculitides show that a panel of biomarkers can successfully discriminate the population groups. The SAFE-T group plans to seek endorsement from health authorities (European Medicines Agency and Food and Drug Administration) to qualify the biomarkers for use in regulatory decision-making processes.
Glucokinase activators (GKAs) are being developed for the treatment of type 2 diabetes. The toxicity of 4 GKAs (PF-04279405, PF-04651887, piragliatin, and PF-04937319) was assessed in mice, rats, dogs, and/or monkeys. GKAs were administered for 2 to 8 weeks. Standard endpoints, glucose, and insulin were assessed. All compounds produced varying degrees of hypoglycemia in all species. Brain neuronal necrosis and/or peripheral neuropathy were observed with most compounds. These findings are consistent with literature reports linking hypoglycemia with nervous system effects. Arteriopathy, mainly of cardiac vessels, was observed at a low frequency in monkey and/or dog. Arteriopathy occurred only at doses that produced severe and prolonged periods of repeated hypoglycemia. Since this lesion occurred in multiple studies with structurally distinct GKAs, these results suggested arteriopathy was related to GKA pharmacology. The morphological characteristics of the arteriopathy were consistent with that produced by experimental catecholamine administration. We hypothesize that the prolonged periods of hypoglycemia resulted in increased local and/or systemic concentrations of catecholamines via a counterregulatory and/or stress-related mechanism. Alternatively, prolonged hypoglycemia may have resulted in endothelial dysfunction leading to arteriopathy. This risk can be managed in human patients in clinical studies by careful glucose monitoring and intervention to avoid prolonged episodes of hypoglycemia.
Male transgenic adenocarcinoma of the mouse prostate (TRAMP) mice are frequently used in prostate cancer research because their prostates consistently develop a series of preneoplastic and neoplastic lesions. Disease progression in TRAMP mouse prostates culminates in metastatic, poorly differentiated carcinomas with neuroendocrine features. The androgen dependence of the rat probasin promoter largely limits transgene expression to the prostatic epithelium. However, extra-prostatic transgene-positive lesions have been described in TRAMP mice, including renal tubuloacinar carcinomas, neuroendocrine carcinomas of the urethra, and phyllodes-like tumors of the seminal vesicle. Here, we describe the histologic and immunohistochemical features of 2 novel extra-prostatic lesions in TRAMP mice: primary anaplastic tumors of uncertain cell origin in the midbrain and poorly differentiated adenocarcinomas of the submandibular salivary gland. These newly characterized tumors apparently result from transgene expression in extra-prostatic locations rather than representing metastatic prostate neoplasms because lesions were identified in both male and female mice and in male TRAMP mice without histologically apparent prostate tumors. In this article, we also calculate the incidences of the urethral carcinomas and renal tubuloacinar carcinomas, further elucidate the biological behavior of the urethral carcinomas, and demonstrate the critical importance of complete necropsies even when evaluating presumably well characterized phenotypes in genetically engineered mice.
Pharmaceutical therapies for non-insulin-dependent diabetes mellitus (NIDDM) include plasma glucose lowering by enhancing glucose utilization. The mitochondrial pyruvate dehydrogenase (PDH) complex is important in controlling the balance between glucose and fatty acid substrate oxidation. Administration of pyruvate dehydrogenase kinase inhibitors (PDHKIs) to rats effectively lowers plasma glucose but results in myocardial steatosis that in some instances is associated primarily with atrial and to a lesser degree with ventricular pathology. Induction of myocardial steatosis is not dose-dependent, varies from minimal to moderate severity, and is either of multifocal or diffuse distribution. Ventricular histopathology was restricted to few myocardial degenerative fibers, while that in the atrium/atria was of either acute or chronic appearance with the former showing myocardial degeneration/necrosis, acute myocarditis, edema, endothelial activation (rounding up), endocarditis, and thrombosis associated with moderate myocardial steatosis and the latter with myocardial loss, replacement fibrosis, and no apparent or minimal association with steatosis. The evidence from these evaluations indicate that excessive intramyocardial accumulation of lipid may be either primarily adverse or represents an indicator of other adversely affected cellular processes.
Over the past decade, laser capture microdissection (LCM) has grown as a tool for gene expression profiling of small numbers of cells from tumor samples and of specific cell populations in complex tissues. LCM can be used to study toxicant effects on selected cell populations within the testis at different stages of spermatogenesis. There are several LCM-related hurdles to overcome, including issues inherent to the method itself, as well as biases that result from amplifying the LCM-isolated RNA. Many technical issues associated with the LCM method are addressed here, including increasing RNA yield and obtaining more accurate quantification of RNA yields. We optimized the LCM method optimized to generate RNA quantities sufficient for quantitative reverse transcription polymerase chain reaction (qRT-PCR) array analysis without amplification and were able to validate the method through direct comparison of results from unamplified and amplified RNA from individual samples. The addition of an amplification step for gene expression studies using LCM RNA resulted in a bias, especially for low abundance transcripts. Although the amplification bias was consistent across samples, researchers should use caution when comparing results generated from amplified and unamplified LCM RNA. Here, we have validated the use of LCM-derived RNA with the qRT-PCR array, improving our ability to investigate cell-type and stage-specific responses to toxicant exposures.
Differences were examined between male and female Sprague-Dawley rats in the response of 16 urinary biomarkers (measured using several assay platforms) to renal injury produced by gentamicin administered subcutaneously for 10 days at a dosage of 75 mg/kg. Urinary biomarkers expressed as fold difference from contemporaneous controls and renal histopathology were assessed after 3 and 10 doses. On day 4, minimal proximal tubular changes were observed microscopically in all males but no females; on day 11, more extensive and more severe injury was observed to a similar extent in all animals of both sexes. Modest increases (maximum 5-fold) in all urinary biomarkers (except epidermal growth factor [EGF], which was decreased) on day 4 and marked elevations (maximum 271-fold) on day 11 were seen consistently in both sexes. However, the magnitude of the increases differed between the sexes. On day 4, despite the lack of tubular injury, many biomarkers were more elevated in females than males but this rarely led to statistically significant sex differences; only 2 biomarkers (β2-microglobulin and total protein) showed a greater increase in males than females in line with the histopathology. On day 11, there were many more biomarkers that showed a statistically significant difference between the sexes in fold change with treatment; in line with the results on day 4, the majority of biomarkers were more increased in females than males. It remains unresolved if sex differences in the magnitude of biomarker response at injury threshold would lead to any difference in diagnostic interpretation between the sexes. These data highlight the need for publication of more studies using animals of both sexes to fully explore the influence of sex on the diagnostic performance of the novel biomarkers.
This investigation examined microRNA-208a (miR-208a) as a potential biomarker of isoproterenol (ISO)-induced cardiac injury in superoxide dismutase-2 (Sod2+/– ) and the wild-type mice, and the potential sensitivity of Sod2+/– mice to ISO-induced toxicity. A single intraperitoneal injection of ISO was administered to age-matched wild-type and Sod2+/– mice at 0, 80, or 160 mg/kg. Plasma miR-208a, cardiac troponin I (cTnI), and ISO systemic exposure were measured at various time points postdose. Hearts were collected for histopathology examination and for tissue expression of miR-208a and myosin heavy chain 7. ISO administration caused increases in cTnI and miR-208a plasma levels that correlated with myocardial damage; however, the magnitude of increase differed according to the types of mice. At similar ISO systemic exposure, the magnitude of cTnI was greater in wild-type mice compared to Sod2+/ – mice; however, the magnitude of miR-208a was greater in Sod2+/– mice than that of the wild-type mice. Myocardial degeneration occurred at ≥3 hr in the wild-type and ≥6 hr in Sod2+/ – mice. At ≥24 hr after ISO administration, miR-208a appeared superior to cTnI in indicating myocardial injury in both wild-type and Sod2+/– mice. Sod2+/– mice were not more sensitive than wild-type mice to ISO-induced toxicity.
The objective of this study was to determine the effects of the phytoestrogen genistein (GEN) on the time of onset and/or the incidence of type 1 diabetes (T1D) in female nonobese diabetic (NOD) mice, when administered GEN by gavage once every day for up to 180 days. Five groups of mice (approximately 24 animals/group; 6–7 weeks of age) were included: naive control, vehicle control (25 mM Na2CO3 in water), and 3 GEN treatment groups (2 mg/kg, 6 mg/kg, and 20 mg/kg). Mice were maintained on a soy- and alfalfa-free diet (5K96) during the study and were monitored for blood glucose changes every week. When compared to the vehicle control, exposure to 2-mg/kg GEN produced significant decreases ranging from 55 to 79% in the total incidences of diabetes (blood glucose ≥ 250 mg/dl) and severe diabetes (blood glucose ≥ 400 mg/dl) starting at week 14 of the study. However, during the later stages of the study (i.e., after week 23), the 2-mg/kg dose had no effect on disease incidence. In animals treated with 6-mg/kg and 20-mg/kg GEN, significant decreases in the total incidence of diabetes were observed starting at week 16, while the incidence of severe diabetes was significantly decreased with the changes being observed initially at weeks 18 and 17 for the 6-mg/kg and 20-mg/kg GEN treatment groups, respectively. Several lines of evidence, including histopathological analysis, suggested that GEN protected the pancreas from autoimmune destruction. However, this protective effect of GEN was absent when female NOD mice were maintained on NTP-2000 rodent diet, which contained 5% soybean meal and 7.5% alfalfa meal (the total concentrations of phytoestrogens ranged between 95 and 134 mg/kg). In summary, oral dosing of GEN reduced the incidence and increased the time to onset of T1D in female NOD mice but only when fed a soy- and alfalfa-free diet.
Differences were examined between male and female Sprague-Dawley rats in basal levels of a wide range of urinary biomarkers, including 7 recently qualified biomarkers. The data were generated from urine samples collected on 3 occasions from untreated rats included in a study of the effect of gentamicin nephrotoxicity on urinary renal biomarkers, reported in a companion article in this journal (Gautier et al. 2014). The performance of multiple assays (9 singleplex assays and 2 multiplex platforms from Rules Based Medicine [RBM] and Meso Scale Discovery [MSD]) was evaluated, and normal ranges and variability estimates were derived. While variability was generally greater on the RBM platform than other assays, the more striking difference in the results from different assays was in magnitude. Where differences were observed between assays for an individual biomarker, they were seen in both sexes and consistent across samples collected at different time points. Differences of up to 15-fold were observed for some biomarker values between assays indicating that results generated using different assays should not be compared. For 8 biomarkers, there was compelling evidence for a sex difference. Baseline values in males were significantly higher than in females for total protein, β2-microglobulin, clusterin, cystatin-C, glutathione-S-transferase (GST-α), tissue inhibitor of metalloproteinases (TIMP-1), and vascular endothelial growth factor (VEGF); female values were significantly higher than that of males for albumin. The largest sex differences (male greater than female by 2- to 11-fold) were seen with β2-microglobulin, GST-α, and TIMP-1. These data add substantially to the limited body of knowledge in this area and provide a useful framework for evaluation of the potential relevance of sex differences in the diagnostic performance of these biomarkers.
Vascular inflammation, infusion reactions, glomerulopathies, and other potentially adverse effects may be observed in laboratory animals, including monkeys, on toxicity studies of therapeutic monoclonal antibodies and recombinant human protein drugs. Histopathologic and immunohistochemical (IHC) evaluation suggests these effects may be mediated by deposition of immune complexes (ICs) containing the drug, endogenous immunoglobulin, and/or complement components in the affected tissues. ICs may be observed in glomerulus, blood vessels, synovium, lung, liver, skin, eye, choroid plexus, or other tissues or bound to neutrophils, monocytes/macrophages, or platelets. IC deposition may activate complement, kinin, and/or coagulation/fibrinolytic pathways and result in a systemic proinflammatory response. IC clearance is biphasic in humans and monkeys (first from plasma to liver and/or spleen, second from liver or spleen). IC deposition/clearance is affected by IC composition, immunomodulation, and/or complement activation. Case studies are presented from toxicity study monkeys or rats and indicate IHC-IC deposition patterns similar to those predicted by experimental studies of IC-mediated reactions to heterologous protein administration to monkeys and other species. The IHC-staining patterns are consistent with findings associated with generalized and localized IC-associated pathology in humans. However, manifestations of immunogenicity in preclinical species are generally not considered predictive to humans.
Previously we found that regulation of eNOS is an important part of the pathogenic process of Drug-induced Vascular Injury (DIVI) for PDE4i. The aims of the current study were to examine the phosphorylation of eNOS in mesentery versus aorta at known regulatory sites across DIVI-inducing drug classes and to compare changes across species. We found that phosphorylation at S615 in rats was elevated 35-fold 2 hr after the last dose of CI-1044 in mesentery versus 3-fold in aorta. Immunoprecipitation studies revealed that many of the upstream regulators of eNOS activation were associated with eNOS in 1 or more signalosome complexes. Next rats were treated with drugs from 4 other classes known to cause DIVI. Each drug was given alone and in combination with SIN-1 (NO donor) or L-NAME (eNOS inhibitor), and the level of eNOS phosphorylation in mesentery and aorta tissue was correlated with the extent of vascular injury and measured serum nitrite. Drugs or combinations produced altered serum nitrite levels as well as vascular injury score in the mesentery. The results suggested that phosphorylation of S615 may be associated with DIVI activity. Studies with the species-specific A2A adenosine agonist CI-947 in rats versus primates showed a similar pattern.
Most published reviews of preclinical toxicological clinical pathology focus on the fundamental aspects of hematology, clinical chemistry, coagulation, and urinalysis in routine toxicology animal species, for example, rats, mice, dogs, and nonhuman primates. The objective of this continuing education course was to present and discuss contemporary examples of nonroutine applications of clinical pathology endpoints used in the drug development setting. Area experts discussed bone turnover markers of laboratory animal species, clinical pathology of pregnant and growing laboratory animals, clinical pathology of nonroutine laboratory animal species, and unique applications of the Siemens Advia® hematology analyzer. This article is a summary based on a presentation given at the 31st Annual Symposium of the Society of Toxicologic Pathology, during the Continuing Education Course titled "Nontraditional Applications of Clinical Pathology in Drug Discovery and Preclinical Toxicology."
The interaction between exposure to nanomaterials and existing inflammatory conditions has not been fully established. Multiwalled carbon nanotubes (MWCNT; Nanocyl NC 7000 CAS no. 7782-42-5; count median diameter in atmosphere 61 ± 5 nm) were tested by inhalation in high Immunoglobulin E (IgE)-responding Brown Norway (BN) rats with trimellitic anhydride (TMA)-induced respiratory allergy. The rats were exposed 2 days/week over a 3.5-week period to a low (11 mg/m3) or a high (22 mg/m3) concentration of MWCNT. Nonallergic animals exposed to MWCNT and unexposed allergic and nonallergic rats served as controls. At the end of the exposure period, the allergic animals were rechallenged with TMA. Histopathological examination of the respiratory tract showed agglomerated/aggregated MWCNT in the lungs and in the lung-draining lymph nodes. Frustrated phagocytosis was observed as incomplete uptake of MWCNT by the alveolar macrophages and clustering of cells around MWCNT. Large MWCNT agglomerates/aggregates were found in granulomas in the allergic rats, suggesting decreased macrophage clearance in allergic rats. In allergic rats, MWCNT exposure decreased serum IgE levels and the number of lymphocytes in bronchoalveolar lavage. In conclusion, MWCNT did not aggravate the acute allergic reaction but modulated the allergy-associated immune response.
A sporadic, diffuse, interstitial mixed cell epididymitis of unknown etiology was noted in the epididymal cauda and distal corpus of young control Sprague-Dawley (SD) rats. Rats from 2 different suppliers were examined as part of routine toxicology studies. The incidence of this finding was 5/5 (study 1), 2/7 (study 2), and 2/7 (study 3). Although 2 of these studies partially overlapped temporally, none of the affected animals from any study was maintained concurrently with affected animals from any of the other 2 studies, and infectious causes, control article toxicity, or autoimmune processes were considered unlikely etiologies. Inflammation similar to that noted in the epididymides of these young rats was not present in other tissues and was not noted in study cohorts sacrificed at ages older than approximately 11 weeks or in rats of similar age from other concurrent studies. Similar findings were noted sporadically in historical control data, and consequently an age-related finding of unknown etiology and occurring in sporadic clusters is reported in SD rats ≤11 weeks old.
Several classes of drugs have been shown to cause drug-induced vascular injury (DIVI) in preclinical toxicity studies. Measurement of blood flow and vessel diameter in numerous vessels and across various tissues by ultrasound imaging has the potential to be a noninvasive translatable biomarker of DIVI. Our objective was to demonstrate the utility of high-frequency ultrasound imaging for measuring changes in vascular function by evaluating blood flow and vessel diameter in the superior mesenteric arteries (SMA) of rats treated with compounds that are known to cause DIVI and are known vasodilators in rat: fenoldopam, CI-1044, and SK&F 95654. Blood flow, vessel diameter, and other parameters were measured in the SMA at 4, 8, and 24 hr after dosing. Mild to moderate perivascular accumulations of mononuclear cells, neutrophils in tunica adventitia, and superficial tunica media as well as multifocal hemorrhage and necrosis in the tunica media were found in animals 24 hr after treatment with fenoldopam and SK&F 95654. Each compound caused marked increases in blood flow and shear stress as early as 4 hr after dosing. These results suggest that ultrasound imaging may constitute a functional correlate for the microscopic finding of DIVI in the rat.
Testicular effects of chemical mixtures may differ from those of the individual chemical constituents. This study assessed the co-exposure effects of the model germ cell- and Sertoli cell-specific toxicants, X-irradiation (x-ray), and 2,5-hexanedione (HD), respectively. In high-dose studies, HD has been shown to attenuate x-ray-induced germ cell apoptosis. Adult rats were exposed to different levels of x-ray (0.5 Gy, 1 Gy, and 2 Gy) or HD (0.33%), either alone or in combination. To assess cell type-specific attenuation of x-ray effects with HD co-exposure, we used laser capture microdissection (LCM) to enrich the targeted cell population and examine a panel of apoptosis-related transcripts using PCR arrays. The apoptosis PCR arrays identified significant dose-dependent treatment effects on several genes, with downregulation of death receptor 5 (DR5), Naip2, Sphk2, Casp7, Aven, Birc3, and upregulation of Fas. The greatest difference in transcript response to exposure was seen with 0.5 Gy x-ray exposure, and the attenuation effect seen with the combined high-dose x-ray and HD did not persist into the low-dose range. Examination of protein levels in staged tubules revealed a significant upregulation in DR5, following high-dose co-exposure. These results provide insight into the testis cell-specific apoptotic response to low-dose co-exposures of model testicular toxicants.
Endoxifen (4-hydroxy-N-desmethyl-tamoxifen), one of the major active metabolites of tamoxifen, has substantially greater estrogen antagonist properties and antiproliferative effects in breast tumor cells than tamoxifen, a mixed estrogen agonist/antagonist. An associated risk of endometrial cancer and hyperplasia has been linked to the estrogen agonist properties of tamoxifen. We evaluated endoxifen using a classic uterotrophic effects method. Rats were given endoxifen or tamoxifen orally for 3 days. Estradiol was the positive control. Endoxifen and tamoxifen plasma levels exceeded those previously observed clinically. Uterine weight was 3-fold higher in the estradiol group than in the tamoxifen or endoxifen groups, which did not differ from vehicle controls. Tamoxifen and endoxifen caused a greater increase in luminal epithelial cell height than estradiol. Both tamoxifen and endoxifen produced an increase in the stromal BrdU labeling index (LI) that was ≤ estradiol and inversely related to dose, but did not affect luminal epithelial cell BrdU LI. As expected, estradiol increased luminal epithelial cell proliferation. These results indicate that endoxifen induces uterotrophic effects, but is less potent than estradiol in eliciting these effects. Given prior preclinical observations that endoxifen has superior antitumor activity than tamoxifen, the observations of similar uterine effects suggest that the endoxifen risk/benefit ratio may be superior to tamoxifen.
The analysis of organ vasculature, and more specifically organ microvasculature, carries special importance for toxicological sciences, and especially for evaluation of drug-induced vascular toxicity. This field presents a special challenge in nonclinical drug safety assessments since there are currently no reliable microvascular toxicity biomarkers. Therefore, we aimed to systematically investigate the use of microvascular 3D geometrical analysis of corrosion casts for evaluation of drug-induced vascular toxicity, utilizing a novel image investigation tool that allows full 3D-quantified geometrical analysis of the entire vascular tree structure. Vascular casts of kidneys from control and low- and high-dose ephedrine/caffeine-treated mice were scanned by a micro CT, and images were processed and analyzed using the VasculomicsTM platform. All evaluations were performed on the kidney cortex. Treatment resulted in a significant and dose-related reduction in overall microvessel density throughout the kidney cortex. This effect was most pronounced for vessels with diameters between 25 µm and 35 µm, and affected mostly vessels located in the superficial part of the kidney cortex. The use of 3D analysis tools in drug-induced vascular toxicity studies allows for very high resolution and characterization of drug effects on the microvasculature and can be used as a valuable tool in drug safety assessments.
The present study describes the normal histology of female reproductive organs during the estrous cycle in the Göttingen minipig. For this purpose, sexually mature females were sacrificed at different phases of the cycle (follicular/proliferation, ovulation, and early-, mid-, and late-luteal/secretory phase). Ovaries, uterus, cervix, vagina, and mammary gland tissues were processed for microscopic evaluation. Sexual maturity was assured by selecting females in which at least 1 progesterone peak was measured. Stage-distinguishing features in ovaries were the Graafian follicles (disrupted vs. nondisrupted) and corpora lutea of recent and preceding cycles (size, cell morphology, and structural composition). In the uterus, stage-specific markers were epithelial morphology, secretory activity (using periodic acid–Schiff/hematoxylin staining), and epithelial mitosis and/or apoptosis. The other reproductive organs were not suitable to discriminate between the different phases of the cycle due to a high morphologic variability (mammary gland, and vagina) or absence of clear morphologic differences between the phases (cervix). The increased use of young minipigs (frequently immature/peripubertal) in preclinical testing requires more knowledge on the histologic cyclic changes. With the present morphologic description of the morphologic characteristics of the reproductive tract in recently ovulating minipigs, a guidance for staging the estrous cycle and determination of sexual immaturity is provided.
The spontaneous incidence of foci of oncocytic proliferation (oncocytic hyperplasia and oncocytoma) was assessed in a histopathological reevaluation of the kidneys of 2,391 male and female Fischer 344 (F344) groups of control rats from long-term carcinogenicity studies (involving 24 chemicals) that had been conducted by the National Toxicology Program. The overall incidence of oncocytic proliferation was 0.3%, with a male preponderance over females at 0.5% (6/1,236) versus 0.09% (1/1,155), respectively. In males, there appeared to be an association of oncocytic proliferation with advanced spontaneous chronic progressive nephropathy. Oncocytoma or oncocytic hyperplasia appear to be rare lesions in F344 rats, and observations from these carcinogenicity studies suggest that they are slow growing and tend to occur late in a rodent’s life span.
Two 6-month repeat-dose toxicity studies in cynomolgus monkeys illustrated immune complex–mediated adverse findings in individual monkeys and identified parameters that potentially signal the onset of immune complex–mediated reactions following administration of RN6G, a monoclonal antibody (mAb). In the first study, 3 monkeys exhibited nondose-dependent severe clinical signs accompanied by decreased erythrocytes with increased reticulocytes, neutrophilia, monocytosis, thrombocytopenia, coagulopathy, decreased albumin, azotemia, and increased serum levels of activated complement products, prompting unscheduled euthanasia. Histologically, immunohistochemical localization of RN6G was associated with monkey immunoglobulin and complement components in glomeruli and other tissues, attributable to immune complex disease (ICD). All 3 animals also had anti-RN6G antibodies and decreased plasma levels of RN6G. Subsequently, an investigational study was designed and conducted with regulatory agency input to detect early onset of ICD and assess reversibility to support further clinical development. Dosing of individual animals ceased when biomarkers of ICD indicated adverse findings. Of the 12 monkeys, 1 developed anti-RN6G antibodies and decreased RN6G exposure that preceded elevations in complement products, interleukin-6, and coagulation parameters and decreases in albumin and fibrinogen. All findings in this monkey, except for antidrug antibody (ADA), reversed after cessation of dosing without progressing to adverse sequelae typically associated with ICD.
Comparative nonclinical studies were conducted with the proposed biosimilar PF-05280586 and rituximab-EU (MabThera®). In side-by-side analyses, peptide maps and complement-dependent cytotoxicity assay results were similar. Sexually-mature cynomolgus monkeys were administered PF-05280586 or rituximab-EU as a single dose of 0, 2, 10, or 20 mg/kg on day 1 and observed for 92 days (single-dose study) or as 5 weekly injections of 0 or 20 mg/kg and necropsied on day 30, the day after the 5th dose, or on day 121 (repeat-dose study). The pharmacokinetic and pharmacodynamic profiles for both molecules were similar. Marked depletion of peripheral blood B cells 4 days after dosing was followed by near or complete repletion (single-dose study) or partial repletion (repeat-dose study). In the single-dose study, anti-drug antibodies (ADA) were detected by day 29 in all animals administered PF-05280586 or rituximab-EU and persisted through day 85, the last day tested. In the repeat-dose study, ADA were detected on day 121 in 50% of animals administered PF-05280586 or rituximab-EU. Both molecules were well tolerated at all doses. In all endpoints evaluated, PF-05280586 exhibited similarity to rituximab-EU.
Pharmacologically, vasoactive agents targeting endothelial and/or smooth muscle cells (SMC) are known to cause acute drug-induced vascular injury (DIVI) and the resulting pathology is due to endothelial cell (EC) perturbation, activation, and/or injury. Alteration in EC structure and/or function may be a critical event in vascular injury and, therefore, evaluation of the circulatory kinetic profile and secretory pattern of EC-specific proteins such as VWF and VWFpp could serve as acute vascular injury biomarkers. In rat and dog models of DIVI, this profile was determined using pharmacologically diverse agents associated with functional stimulation/perturbation (DDAVP), pathological activation (lipopolysaccharide [LPS]/endotoxin), and structural damage (fenoldopam [FD], dopamine [DA], and potassium channel opener (PCO) ZD6169). In rats, FD caused moderate DIVI and time-related increase in plasma VWF levels ~33% while in control rats VWF increased ~5%. In dogs, VWF levels transiently increased ~30% when there was morphologic evidence of DIVI by DA or ZD6169. However, in dogs, VWFpp increased >60-fold (LPS) and >6-fold (DDAVP), respectively. This was in comparison to smaller dynamic 1.38-fold (LPS) and 0.54-fold (DDAVP) increases seen in plasma VWF. Furthermore, DA was associated with a dose-dependent increase in plasma VWFpp. In summary, VWF and VWFpp can discriminate between physiological and pathological perturbation, activation, and injury to ECs.
The incidence and range of spontaneous central nervous system tumors were determined in control Charles River rodents (Sprague-Dawley, Han-Wistar, Wistar rats, and CD-1 mice) from regulatory carcinogenicity studies carried out over the period 2002 to 2013 and were compared with the previously published data. In both species, the brain was notably more affected than the spinal cord. Incidences were comparable overall between rat strains (2.33%, 2.54%, and 2.89% in Wistar, Sprague-Dawley, and Han-Wistar strains, respectively) and were low in CD-1 mice (0.42% in 104-week studies and 0.2% in 80-week studies). Predominant tumor types were granular cell tumors in Wistar and Han-Wistar rats and malignant astrocytoma in Sprague-Dawley rats. Male rats were more frequently affected than females, but no sex predilection was apparent in CD-1 mice. Occasional early-onset tumors were diagnosed in rats from study week 23 onward. It is hoped that these results will provide the pathologist and the toxicologist with an up-to-date database of background neoplastic findings in widely used rodent strains.
Vascular endothelial growth factor (VEGF) receptor tyrosine kinase (RTK) inhibitors are reported to cause reversible mucosal hyperplasia (adenosis) in the duodenum of rats; however, the pathogenesis is not fully elucidated. Using lenvatinib, a VEGF RTK inhibitor, we characterized the histologic time course of this duodenal change in rats. At 4 weeks, there was degeneration and necrosis of Brunner’s gland epithelium accompanied by neutrophil infiltration around the affected glands. At 13 weeks, the inflammation was more extensive, and Brunner’s gland epithelium was attenuated and flattened and was accompanied by reactive hyperplasia of duodenal epithelium. At 26 weeks, the changes became more severe and chronic and characterized by marked cystic dilation, which extended to the external muscular layer. These dilated glands exhibited morphological characteristics of duodenal crypt epithelium, suggestive of replacement of disappeared Brunner’s glands by regenerative duodenal crypt epithelial cells. Similar changes were not present in similar time course studies in dog and monkey studies, suggesting that this is a rodent- or species-specific change. Based on the temporal progression of Brunner’s gland lesion, we identify degeneration and necrosis of the Brunner’s glands as the primary change leading to inflammation, cystic dilatation, and regeneration with cells that are morphologically suggestive of duodenal crypt epithelium.
A new service to facilitate career advancement opportunities has been implemented by the American College of Veterinary Pathologists (ACVP)/Society of Toxicologic Pathology (STP) Coalition for Veterinary Pathology Fellows. This service will allow rapid communication of these opportunities between veterinary pathologists in academia, industry, and government, and will be useful to trainees as well as established pathologists.
The results of repeat-dose toxicity tests are usually presented as tables of means and standard deviations (SDs), with an indication of statistical significance for each biomarker. Interpretation is based mainly on the pattern of statistical significance rather than the magnitude of any response. Multiple statistical testing of many biomarkers leads to false-positive results and, with the exception of growth data, few graphical methods for showing the results are available. By converting means and SDs to standardized effect sizes, a range of graphical techniques including dot plots, line plots, box plots, and quantile–quantile plots become available to show the patterns of response. A bootstrap statistical test involving all biomarkers is proposed to compare the magnitudes of the response between treated groups. These methods are proposed as an extension rather than an alternative to current statistical analyses. They can be applied to published work retrospectively, as all that is required is tables of means and SDs. The methods are illustrated using published articles, where the results range from strong positive to completely negative responses to the test substances.
In a 2-year rat carcinogenicity study, pegvisomant injected subcutaneously on a daily basis at doses of 0, 2, 8, or 20 mg/kg/day produced malignant fibrous histiocytomas (MFHs) at the injection sites of 3 male rats (5%) given 8 mg/kg/day and 5 males (8%) given 20 mg/kg/day. MFH was characterized by unencapsulated dermal and subcutaneous sheets of fusiform and spindle-shaped cells sometimes with areas of round and/or irregular, pleomorphic cells and variable numbers of large multinucleated giant cells. Some regions of MFH had a fibroblastic appearance with streaming cells forming storiform patterns, while other areas consisted primarily of round to plump irregular cells with more giant cells. Pegvisomant did not increase the incidence of MFH in female rats and did not produce any other neoplastic responses in rats. In the dermis and subcutis at the injection sites of many males and females, pegvisomant produced dose-related increased incidences and severity of histiocytic infiltrates consisting of vacuolated macrophages with variable mature or immature fibrous tissue. Neoplasms at injection sites did not result in marketing restrictions or a label warning for human cancer risk, highlighting that injection-site neoplasms in rats have low relevance for human risk assessment.
The purpose of this article is to characterize skin lesions in cynomolgus monkeys following vildagliptin (dipeptidyl peptidase-4 inhibitor) treatment. Oral vildagliptin administration caused dose-dependent and reversible blister formation, peeling and flaking skin, erosions, ulcerations, scabs, and sores involving the extremities at ≥5 mg/kg/day and necrosis of the tail and the pinnae at ≥80 mg/kg/day after 3 weeks of treatment. At the affected sites, the media and the endothelium of dermal arterioles showed hypertrophy/hyperplasia. Skin lesion formation was prevented by elevating ambient temperature. Vildagliptin treatment also produced an increase in blood pressure and heart rate likely via increased sympathetic tone. Following treatment with vildagliptin at 80 mg/kg/day, the recovery time after lowering the temperature in the feet of monkeys and inducing cold stress was prolonged. Ex vivo investigations showed that small digital arteries from skin biopsies of vildagliptin-treated monkeys exhibited an increase in neuropeptide Y–induced vasoconstriction. This finding correlated with a specific increase in NPY and in NPY1 receptors observed in the skin of vildagliptin-treated monkeys. Present data provide evidence that skin effects in monkeys are of vascular origin and that the effects on the NPY system in combination with increased peripheral sympathetic tone play an important pathomechanistic role in the pathogenesis of cutaneous toxicity.
One of the biggest challenges in drug development is interpretation of findings suggestive of immunostimulation, particularly when the findings are unexpected based on the known mechanism of action of the drug. Findings suggestive of immunostimulation do not necessarily preclude development of the drug. By understanding mechanism, the patient population, and species differences and by careful clinical monitoring, many of the drugs that produce immunostimulation can be successfully developed. This opinion piece describes various types of immunostimulation and a risk assessment strategy for management of unexpected immunostimulation in toxicity studies.
The mechanistic relationship between increased food consumption, increased body weights, and increased incidence of tumors has been well established in 2-year rodent models. Body weight parameters such as initial body weights, terminal body weights, food consumption, and the body weight gains in grams and percentages were analyzed to determine whether such relationship exists between these parameters with the incidence of common spontaneous tumors in Tg.rasH2 mice. None of these body weight parameters had any statistically significant relationship with the incidence of common spontaneous tumors in Tg.rasH2 males, namely lung tumors, splenic hemangiosarcomas, nonsplenic hemangiosarcomas, combined incidence of all hemangiosarcomas, and Harderian gland tumors. These parameters also did not have any statistically significant relationship with the incidence of lung and Harderian gland tumors in females. However, in females, increased initial body weights did have a statistically significant relationship with the nonsplenic hemangiosarcomas, and increased terminal body weights did have a statistically significant relationship with the incidence of splenic hemangiosarcomas, nonsplenic hemangiosarcomas, and the combined incidence of all hemangiosarcomas. In addition, increased body weight gains in grams and percentages had a statistically significant relationship with the combined incidence of all hemangiosarcomas in females, but not separately with splenic and nonsplenic hemangiosarcomas.
In a 13-week feeding toxicity study of grape skin extract (GSE) performed previously, 5.0% GSE showed diffuse hypertrophy and basophilia in rat parotid glands. To clarify whether the change in the parotid glands was an adverse effect of GSE, 6-week-old male F344 rats were fed a diet containing 5.0% GSE or were administered a dose corresponding to the dietary concentration via gavage for 4 weeks, and the treatment was stopped for 2 weeks. To ascertain the effect of astringency, other animals were fed a diet containing 5.0% tannic acid (TA) using the same protocol as the GSE feed group. Control groups were fed a basal diet or were administered sterilized distilled water by gavage. In the GSE and TA feed groups, diffuse severe hypertrophy and basophilia in the parotid glandular epithelial cells were observed. Macroscopic, microscopic, and ultrastructural characteristics consistent with cellular hypertrophy was less apparent after the recovery period in both feed groups. In contrast, no changes were observed in the parotid glands of the gavage GSE and control groups at week 4. Based on these findings of parotid hypertrophy without cytotoxicity, the data from this and previous studies suggest that hypertrophy of the parotid glands induced by feeding treatment with GSE is an adaptive non-adverse effect that is reversible upon removal of the sialotrophic agent.
The aim of this retrospective study was to assess the incidence and severity of tubular atrophy/hypoplasia in the testes of 104 control Göttingen minipigs aged 4.5 to 15 months. The finding was termed "tubular hypoplasia/atrophy" according to published descriptions for the dog. It included different microscopic changes that were considered part of a continuum, namely seminiferous epithelium vacuolation, presence of multinucleated germ cells in the tubular lumen, and decreased numbers (hypospermatogenesis) or absence of germ cells. This retrospective study demonstrates that tubular hypoplasia/atrophy is present in more than 70% of Göttingen minipigs and can occur at a marked severity in control animals. It correlated with a higher level of cell debris and a decrease in sperm content in the epididymides and with lower absolute and relative testes weights. There was no correlation with the weight of other sexual organs, total bodyweight, or age, which demonstrates that this change was not related to sexual immaturity. The distinction between this background finding and a compound-related effect could be challenging for the pathologist.
Chronic administration of drisapersen, a 2'-OMe phosphorothioate antisense oligonucleotide (AON) to mice and monkeys resulted in renal tubular accumulation, with secondary tubular degeneration. Glomerulopathy occurred in both species with species-specific characteristics. Glomerular lesions in mice were characterized by progressive hyaline matrix accumulation, accompanied by the presence of renal amyloid and with subsequent papillary necrosis. Early changes involved glomerular endothelial hypertrophy and degeneration, but the chronic glomerular amyloid and hyaline alterations in mice appeared to be species specific. An immune-mediated mechanism for the glomerular lesions in mice was supported by early inflammatory changes including increased expression of inflammatory cytokines and other immunomodulatory genes within the renal cortex, increased stimulation of CD68 protein, and systemic elevation of monocyte chemotactic protein 1. In contrast, kidneys from monkeys given drisapersen chronically showed less severe glomerular changes characterized by increased mesangial and inflammatory cells, endothelial cell hypertrophy, and subepithelial and membranous electron-dense deposits, with ultrastructural and immunohistochemical characteristics of complement and complement-related fragments. Lesions in monkeys resembled typical features of C3 glomerulopathy, a condition described in man and experimental animals to be linked to dysregulation of the alternative complement pathway. Thus, inflammatory/immune mechanisms appear critical to glomerular injury with species-specific sensitivities for mouse and monkey. The lower observed proinflammatory activity in humans as compared to mice and monkeys may reflect a lower risk of glomerular injury in patients receiving AON therapy.
Osteoporosis is a very common skeletal disorder characterized by reduced bone mass and altered trabecular microarchitecture that leads to bone fragility and fractures. Such disease is due to alterations of the remodeling process that occurs in the basic multicellular units that are transitory cellular complexes including an osteoclastic phase (osteoclast activation and resorption of microscopic portions of bone), a reversion phase (osteoclast replacement by so-called postosteoclastic cells), and an osteoblastic phase (osteoblastic reconstruction of the resorbed bone matrix till the initial volume is regained). Bone remodeling is regulated by a number of systemic and local factors; among the former, besides physical activity and mechanical stresses, a primary role is played by hormones such as parathyroid hormone, vitamin D metabolites, estrogens, calcitonin, and glucocorticoids; among the latter, several growth factors (macrophage colony-stimulating factor, transforming growth factor β, platelet-derived growth factor, fibroblast growth factor 1, bone morphogenetic protein, and insulin-like growth factor 1), as well as the osteoprotegerin-receptor activator of nuclear factor- B ligand system and the sclerostin, play a primary function. The remodeling phases can be evaluated by static and dynamic histomorphometry. Their abnormalities may lead to several osteopathies, the most common of which is osteoporosis (above all senile and postmenopausal), a rather elusive disease chiefly due to its slow development. The use of animal models in its study is emphasized.
Histopathology generally represents the reference standard for performance evaluation of nonclinical biomarkers used to inform regulatory decision making. This study uses drug-induced nephrotoxicity in rats to evaluate histopathology methods utilized in biomarker performance assessments. Male Sprague-Dawley rats received a single dose of cisplatin (0.5–5.0 mg/kg, intraperitoneally) to produce mild renal injury. Animals were euthanized 72 hr postdose and perfusion fixed. Kidneys were processed for histology and stereology procedures. Kidney injury molecule-1 (Kim-1) was measured in urine and in kidney tissue. Digital slide images were generated and analyzed by pathologists after collaborating on a training set of glass slides and digital images. Image analysis identified immunohistochemistry (IHC)-defined tubular injury. Stereology methods yielded estimations of proximal tubular cell number and volume. Statistical relationships among data sets were determined using correlation coefficients. Receiver operator characteristic (ROC) analyses determined the effect of method on biomarker assessment. Urinary Kim-1 was strongly correlated with digital image analysis and secondarily to histopathology evaluations. Stereology demonstrated weak or no correlation to pathology and urinary Kim-1. In ROC analyses, semiquantitative evaluations determined higher values for urinary Kim-1 performance than did IHC-based qualitative digital analyses. Semiquantitative evaluation as used in this study was most predictive of urinary Kim-1 values.
In preclinical studies of pharmaceutical agents, the beagle dog is a commonly used model for the detection of cardiotoxicity. Incidental findings, postmortem changes, and artifacts must be distinguished histopathologically from test item–related findings in the heart. In this retrospective analysis, cardiac sections from 88 control beagles (41 male, 47 female; ages 5–18 months) in preclinical studies were examined histopathologically. The most common finding was thickening of the tunica media of intramural coronary arteries, most likely a postmortem change. The second most common finding was the presence of vacuoles within Purkinje fibers. Dilated lymphatic and blood vessels at the insertion of chordae tendineae were noted more commonly in males than in females and were considered a normal anatomic feature. Mesothelial-lined papillary fronds along the epicardial surface of the atria were present in several dogs, as were small infiltrates of inflammatory cells usually within the myocardium. In summary, control beagles’ hearts frequently have incidental findings that must be differentiated from test item–related pathologic changes. Historical control data can be useful for the interpretation of incidental and test item–related findings in the beagle heart.
Understanding the long-term effects and possible toxicity of nanoceria, a widely utilized commercial metal oxide, is of particular importance as it is poised for development as a therapeutic agent based on its autocatalytic redox behavior. We show here evidence of acute and subacute adverse hepatic responses, after a single infusion of an aqueous dispersion of 85 mg/kg, 30 nm nanoceria into Sprague Dawley rats. Light and electron microscopic evidence of avid uptake of nanoceria by Kupffer cells was detected as early as 1 hr after infusion. Biopersistent nanoceria stimulated cluster of differentiation 3+ lymphocyte proliferation that intermingled with nanoceria-containing Kupffer cells to form granulomata that were observed between days 30 and 90. Ultrastructural tracking of ceria nanoparticles revealed aggregated nanoceria in phagolysosomes. An increased formation of small nanoceria over time observed in the latter suggests possible dissolution and precipitation of nanoceria. However, the pathway for nanoceria metabolism/secretion remains unclear. Although frank hepatic necrosis was not observed, the retention of nanoceria increased hepatic apoptosis acutely, this persisted to day 90. These findings, together with our earlier reports of 5-nm ceria-induced liver toxicity, provide additional guidance for nanoceria development as a therapeutic agent and for its risk assessment.
(+)-Usnic acid (UA) has been known to be a strong uncoupler, and mitochondrial and endoplasmic reticulum (ER)–related stresses are suggested to be involved in the mechanism of hepatotoxicity. However, it has not been clarified whether UA causes toxicity in other mitochondria-rich organs such as the heart. We elucidated whether UA induces cardiotoxicity and its mechanism. UA was orally administered to rats for 14 days, and laboratory and histopathological examinations were performed in conjunction with toxicogenomic analysis. As a result, there was no alteration in blood chemistry, whereas cytoplasmic rarefaction of myocardium was observed microscopically. This finding corresponded to the swollen mitochondria observed ultrastructurally. Immunohistochemically, expression of prohibitin, indicating mitochondrial imbalance, increased in the sarcoplasmic area. Toxicogenomic analysis highlighted the upregulation of gene groups consisting of oxidative stress, ER stress, and amino acid limitation. Interestingly, the number of upregulated genes was larger in the amino acid limitation-related gene group than that in other groups, implying that amino acid limitation might be one of the sources of oxidative stress, not only mitochondria and ER-originated stresses. In conclusion, the heart was manifested to be one of the target organs of UA. Mitochondrial imbalance with complex stresses may be involved in the toxic mechanism.
Lersivirine is a nonnucleoside reverse transcriptase inhibitor (NNRTI) being developed for the treatment of HIV-1 infection. Like other NNRTIs, lersivirine is a potent enzyme inducer in rodents capable of inducing a number of hepatic enzymes including those involved in its own metabolism. Preclinically lersivirine has been associated with hepatocellular hypertrophy and thyroid gland follicular cell hypertrophy in rats, mice, and dogs. In rodents, we show that development of thyroid hypertrophy is related to the classic mechanism, namely increased thyroxine (T4) clearance secondary to induction of uridine-diphosphoglucuronosyltransferase (UDPGT) in the liver and a resulting increase in thyroid-stimulating hormone. Similarly, lersivirine-exposed dogs exhibit a significant increase in hepatic UDPGT enzyme activity along with increased T4 clearance although clear effects on serum thyroid hormone levels were less apparent. These effects on thyroid hormonal clearance in the dog suggest that thyroid gland hypertrophy in this species is due to the same mechanism shown to occur in rodents although, as expected, dogs better adapt to these effects and therefore maintain relatively normal thyroid hormonal balance. It is also notable that the minimal thyroid follicular hypertrophy that occurs in dogs does not progress as is seen in rodents. As is the case with rodents, these adaptive changes in the dog are not considered indicative of a human health risk.
Cardiac troponins serve as serum biomarkers of myocardial injury. The current study examined the influence of age on serum concentrations of cardiac troponin I (cTnI). An ultrasensitive immunoassay was used to monitor cTnI concentrations in Sprague-Dawley (SD) rats and Erythrocebus patas monkeys of different ages. The mean cTnI concentrations were highest in 10-day-old rats compared to 25-, 40-, and 80-day-old SD rats. Cardiomyocyte remodeling was apparent in hearts from 10-day-old SD rats as evident by hypercellularity, irregularly shaped nuclei, and moderate numbers of myocytes undergoing mitosis and apoptosis. The mean concentration of cTnI in 5 newborn monkeys was considerably higher than that of three 1-year-old monkeys. Evidence of cardiomyocyte remodeling was also observed in these newborn hearts (loss of myofibrils and cytoplasmic vacuolation). Commercial animal serum samples were also analyzed. The concentrations of cTnI detected in fetal equine and porcine serum were considerably higher than that found in adult equine and porcine serum samples Likewise, fetal bovine serum had higher cTnI concentrations (>2,400 pg/ml) than did adult caprine and laprine samples (2.5–2.7 pg/ml). The present study found age-related differences in cTnI concentrations, with higher levels occurring at younger ages. This effect was consistent across several animal species.
The development process of myelination varies between region and species. Fully myelinated fibers are required if mammalian neural circuits are to function normally. Histology samples at staggered time points throughout the study were examined at days 4, 5, 7, 8, 10, 14, 17, 24, 37, and 44. We suggest that the development of myelin in the juvenile rodent brain can be conveniently separated into 3 phases. Evaluation of myelin basic protein–stained sections of the areas of brain that contain the elements of the developing limbic system over the sensitive period from postnatal day (PND) 14 to 34 may provide an insight into possible toxicity that may lead to cognition and learning issues in adults. We will hope to develop this notion further in the future. The precise chronology of the development of the blood–brain barrier in rats has yet to be established; thus, there is potential for significant exposure of the juvenile brain to chemicals that do not cross the blood–brain barrier in the adult. Thus, it is suggested that evaluation of myelin development should probably be extended to all new chemical entities intended for pediatric use, and not just those that are intended for central nervous system use.
In this study, histopathologic changes following carbaryl exposure for 96 hr were investigated in the digestive tract of Levantine frog, Pelophylax bedriagae. Adult frogs were exposed to carbaryl once by oral gavage in concentrations of 0.05, 0.1, and 0.2 mg/g. Histopathological changes were more prominent in medium- (0.1 mg/g) and high-dose (0.2 mg/g) groups than in the low-dose (0.05 mg/g) group. Esophageal cells showed vacuolization, cellular swelling, nuclear pyknosis, karyolysis, and necrosis. Additionally, esophageal glandular atrophy and infiltration of inflammatory cells around esophageal glands were observed at medium and high doses. In the stomach, there were prominent histopathologic defects such as cellular swelling and necrosis in gastric glands, necrotic cells within the interstitial spaces, separation of epithelial cell layer, congested vessels, and hemorrhage at medium and high doses. In the intestine, detachment of epithelial layer, epithelial cell disorganization, inflammation, and necrosis were detected at medium and high doses. The results of this study showed that carbaryl caused adverse effects on the digestive tract of the Levantine frog, P. bedriagae.
Antiretroviral therapy has dramatically reduced mortality in human immunodeficiency virus (HIV) infection. In 1988, the suggestion that the first antiretroviral drug, zidovudine, was the potential cause of muscle pathology in HIV-infected persons resulted in structural and biochemical patient studies demonstrating acquired mitochondrial dysfunction. Assessment of subsequent nucleoside analog reverse transcriptase inhibitor (NRTI) antiretroviral drugs has indicated that mitochondria are a common target of NRTI toxicity in multiple tissues, leading to a wide variety of pathology ranging from lipodystrophy to neuropathy. Overwhelmingly, these complications have emerged during post-licensing human studies. Subsequent animal and in vitro studies have then elucidated the potential pathological mechanisms, suggesting that NRTI-associated mitochondrial toxicity arises principally from inhibition of the sole mitochondrial DNA (mtDNA) polymerase gamma, leading to a reduction in mtDNA content (depletion). Millions of patients have been treated with mitochondrially toxic NRTIs and these drugs remain the backbone of antiretroviral rollout in much of sub-Saharan Africa. Here we describe the 25-year history of antiretroviral associated mitochondrial pathology and critically review the strength of evidence linking clinical, histopathological, and molecular data. We discuss recently described novel mechanisms of NRTI-associated mitochondrial damage and whether or not recently licensed NRTIs may be considered free from mitochondrial toxicity.
Estrogens and androgens affect male and female reproductive systems. Recently, we reported that prenatal di(n-butyl) phthalate (DBP) exposure induced atypical Leydig cells (LCs) hyperplasia during adulthood. The present study investigated the expression of estrogen receptor α (ERα), estrogen receptor β (ERβ), and androgen receptor (AR) in LCs of 5-, 7-, 9-, 14-, and 17-week-old Sprague-Dawley (srl) rats whose dams had been administered DBP intragastrically at 100 mg/kg/day or the vehicle (corn oil) from days 12 to 21 postconception. Immunohistochemical, Western blotting, and reverse transcription polymerase chain reaction analyses revealed that the expressions of ERα, ERβ, and AR proteins and mRNAs in the DBP group were similar to those of the vehicle group at 5 and 7 weeks, but significantly higher ERα and lower ERβ and AR levels were observed in the DBP group at 9 to 17 weeks. The rats prenatally exposed to DBP had seminiferous tubule degeneration and atypical hyperplasia of LCs during adulthood, which was associated with an increase in expression of ERα and a decrease of ERβ and AR in the testis.
Thionamides such as propylthiouracil (PTU) and methimazole (MMI) have been used for more than 50 years to treat the more common causes of thyrotoxicosis/hyperthyroidism such as Graves’ disease. Serious adverse effects associated with thionamides in humans include idiosyncratic liver damage, agranulocytosis, aplastic anemia, and vasculitis. Both prospective and retrospective clinical studies with these drugs have failed to identify predictive biomarker for these adverse effects. To assess whether rat is a good model for predicting drug-related adverse events in the liver and in the bone marrow, we conducted a comprehensive study in male rats with multiple doses of PTU and MMI. As expected, euthyroid animals became hypothyroid along with several secondary changes associated with hypothyroidism. There were slight reductions in red blood cell parameters along with some marginal effects on the bone marrow elements. However, there was no evidence of significant neutropenia and liver injury in both PTU-treated and MMI-treated cohorts. MMI-related effects were noted in the seminiferous tubules of the testes. Overall, 1-month daily treatment of euthyroid rats with PTU or MMI resulted in hypothyroidism, minor bone marrow effects, and several secondary effects associated with hypothyroidism, but without any evidence of adverse effects reported in humans including liver injury and agranulocytosis.
Aged male Fischer 344/N rats are prone to developing spontaneous peritoneal mesotheliomas that arise predominantly from the tunica vaginalis of the testes. A definitive cause for the predominance of this neoplasm in F344/N rats is unknown. Investigation of the molecular alterations that occur in spontaneous rat mesotheliomas may provide insight into their pathogenesis as well enable a better understanding regarding the mechanisms underlying chemically induced mesothelioma in rodents. Mesothelial cell function represents a complex interplay of pathways related to host defense mechanisms and maintenance of cellular homeostasis. Global gene expression profiles of spontaneous mesotheliomas from vehicle control male F344/N rats from 2-year National Toxicology Program carcinogenicity bioassays were analyzed to determine the molecular features of these tumors and elucidate tumor-specific gene expression profiles. The resulting gene expression pattern showed that spontaneous mesotheliomas are associated with upregulation of various growth factors, oncogenes, cytokines, pattern recognition response receptors, and pathogen-associated molecular patterns receptors, and the production of reactive oxygen and nitrogen species, as well as downregulation of apoptosis pathways. Alterations in these pathways in turn trigger molecular responses that stimulate cell proliferation and promote tumor survival and progression.
International regulatory and pharmaceutical industry scientists are discussing revision of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) S1 guidance on rodent carcinogenicity assessment of small molecule pharmaceuticals. A weight-of-evidence approach is proposed to determine the need for rodent carcinogenicity studies. For compounds with high human cancer risk, the product may be labeled appropriately without conducting rodent carcinogenicity studies. For compounds with minimal cancer risk, only a 6-month transgenic mouse study (rasH2 mouse or p53+/– mouse) or a 2-year mouse study would be needed. If rodent carcinogenicity testing may add significant value to cancer risk assessment, a 2-year rat study and either a 6-month transgenic mouse or a 2-year mouse study is appropriate. In many cases, therefore, one rodent carcinogenicity study could be sufficient. The rasH2 model predicts neoplastic findings relevant to human cancer risk assessment as well as 2-year rodent models, produces fewer irrelevant neoplastic outcomes, and often will be preferable to a 2-year rodent study. Before revising ICH S1 guidance, a prospective evaluation will be conducted to test the proposed weight-of-evidence approach. This evaluation offers an opportunity for a secondary analysis comparing the value of alternative mouse models and 2-year rodent studies in the proposed ICH S1 weight-of-evidence approach for human cancer risk assessment.
A typical 26-week Tg.rasH2 mouse carcinogenicity study usually has 4 dose groups, composed of 25 mice/sex, which include 1 control and 3 test article–treated groups. In every study, there is a protocol required full tissue list of 49 tissues which is examined microscopically in all animals of these 4 dose groups. Based on retrospective analysis of the historical control data collected from studies conducted in Tg.rasH2 mice from 2004 to 2012, we propose that a full tissue list be examined as usual in the control and high-dose groups; however, in the low- and mid-dose groups, only select tissues should be examined. The select tissue list is generated after analyzing common tumors, uncommon tumors seen grossly, uncommon tumors not seen grossly, organ weight variations with accountable microscopic lesions, and target organs identified in the high-dose groups. The proposed changes to the International Conference on Harmonization S1 guidance may lead to an increased need for 26-week Tg.rasH2 mice studies. The time savings resulting from processing and evaluating a select tissue list rather than a full tissue list from low- and mid-dose groups of Tg.rasH2 mouse studies will further accelerate early completion of these studies without compromising the quality and integrity.
Whether biliary proliferative lesions in nonclinical species are predictive of potential hepatotoxicity in humans depends, at least in part, on the nature and severity of such changes in the nonclinical species. We reviewed published literature (clinical and nonclinical) and experimental data from rat toxicology studies conducted by GlaxoSmithKline and the National Institute of Environmental Health Sciences’ National Toxicology Program in an effort to better characterize the relative risk of hepatobiliary effects in humans. Available evidence supports the interpretation that minimal "typical" appearing bile duct hyperplasia limited to the portal triads may be considered non-adverse in the rat and is of little to no concern to humans. The toxicological relevance of mild to moderate "typical" hyperplasia is less certain, and may be considered adverse in the rat and potentially pose a risk for humans, particularly if accompanied by evidence of hepatobiliary injury or functional compromise. In addition, any proliferative lesion that includes atypical or dysplastic epithelial changes, oval cell proliferation, and/or significant extension beyond the portal tracts is considered more ominous and may be considered adverse in the rat.
Ginkgo biloba extract (GBE) is a popular herbal supplement that is used to improve circulation and brain function. In spite of widespread human exposure to relatively high doses over potentially long periods of time, there is a paucity of data from animal studies regarding the toxicity and carcinogenicity associated with GBE. In order to fill this knowledge gap, 3-month and 2-year toxicity and carcinogenicity studies with GBE administered by oral gavage to B6C3F1/N mice and F344/N rats were performed as part of the National Toxicology Program’s Dietary Supplements and Herbal Medicines Initiative. The targets of GBE treatment were the liver, thyroid, and nose. These targets were consistent across exposure period, sex, and species, albeit with varying degrees of effect observed among studies. Key findings included a notably high incidence of hepatoblastomas in male and female mice and evidence of carcinogenic potential in the thyroid gland of both mice and rats. Various nonneoplastic lesions were observed beyond control levels in the liver, thyroid gland, and nose of rats and mice administered GBE. Although these results cannot be directly extrapolated to humans, the findings fill an important data gap in assessing risk associated with GBE use.
Transgenic organisms that express fluorescent proteins are used frequently for in vivo visualization of proteins and cells. The phenotype of a transgenic medaka (Oryzias latipes) strain that expresses a red fluorescent protein (RFP) in hepatocytes was characterized using light and fluorescence microscopy, immunohistochemistry, and transmission electron microscopy (TEM). Expression of RFP was first detected by confocal fluorescence microscopy in the location of the liver bud of live medaka embryos at 60 hr postfertilization (developmental stage 27). Subsequently, RFP signal was observed exclusively in hepatocytes throughout life using fluorescence microscopy in live fish and immunohistochemistry in formalin-fixed, paraffin-embedded liver sections. As the fish aged, prominent intracytoplasmic eosinophilic inclusions immunoreactive for RFP were observed by light microscopy and were correlated with membrane-bound electron dense inclusions on TEM. These results define the onset and location of RFP expression in the Tg(zf.L-fabp:DsRed) medaka and characterize a histologic phenotype that results from RFP expression in hepatocytes.
The advancement of technology and the growth of international commerce underscore the need for global harmonization of regulatory safety requirements and their assessment pertaining to consumer products such as drugs, medical devices, and food. This need is particularly relevant when safety requirements involve time-intensive and costly animal safety studies. Here we present the current regulatory requirements in Europe, the United States, and Japan for flavoring substances (FSs) used in foods and point out significant differences relevant to the international standardization for safety assessments that in our opinion need to be addressed and overcome. The safety assessments that are carried out for FSs in various countries are influenced by divergent definitions of FS, by the information required and available for regulatory submission, and by different regulatory procedures, including the use of decision tree approaches. The European Food Safety Authority (EFSA), the Expert Panel of the U.S. Flavor and Extract Manufacturers Association (FEMA), and the Joint Food and Agriculture Organization (FAO)/World Health Organization (WHO) Expert Committee on Food Additives (JECFA) are making efforts to improve and harmonize the safety assessment of FSs. The application of in silico methods such as quantitative structure–activity relationships and read-across strategies relying on expert input are useful as a first-step screening of the assessment. Application of the Threshold of Toxicological Concern (TTC) approach permits conclusions that are compatible with the risk assessment approaches currently used by international advisory committees.
The Japanese Regulatory Authority, on the other hand, does not yet consider in silico methods but still requires in vivo and in vitro genotoxicity test data as well as repeat-dose 90-day toxicity data in at least one species, to be submitted as the first step in the safety assessment of FSs. With this article, we echo requests that have been made for xenobiotics by the pharmaceutical industry worldwide, extending them to food-related products, especially FSs. We encourage regulatory agencies to adopt globally harmonized safety assessment procedures, regulatory guidelines, and review practices for FSs to foster global trade and to reduce costs and laboratory animal use.
Previous reports investigating the mechanisms of galactosamine toxicity have discussed the presence of responders and nonresponders after intraperitoneal (IP) administration of a toxic dose. The incidence of nonresponders has been reported to be as high as 47%. To rule out inadvertent intestinal, solid organ, or subcutaneous injection as at least a partial cause for the variability, we performed midline incisions and dosed 10 rats via a flexible catheter, with a toxic dose of galactosamine. Results were compared to a previous range finding study with IP-injected rats. As opposed to the IP-injected rats that had a roughly 50% response rate (based on serum alanine aminotransferase [ALT] elevation) and 100% of the midline incision catheter-instilled rats had elevations in ALT. Saline controls had no elevations. Histopathologic examination of livers from 5 midline-incisioned rats euthanized 48 hr after dosing with the lowest ALT responses revealed portal eosinophilic infiltrates and biliary hypertrophy/hyperplasia contiguous with areas of necrosis. Examination of 5 rats with the highest ALT elevations euthanized 10 days post dose revealed similar lesions to be resolving. We conclude that a significant contribution to variability in response to IP-injected galactosamine and possibly other investigative drugs is inadvertent misinjection of all or part of the dose.
Because of the potential for exposure to N,N-dimethyl-p-toluidine (DMPT) in medical devices and the lack of toxicity and carcinogenicity information available in the literature, the National Toxicology Program conducted toxicity and carcinogenicity studies of DMPT in male and female F344/N rats and B6C3F1/N mice. In these studies, a treatment-related macrocytic regenerative anemia characterized by increased levels of methemoglobin and Heinz body formation developed within a few weeks of DMPT exposure in rats and mice. DMPT induced nasal cavity, splenic, and liver toxicity in rats and mice at 3 months and 2 years. DMPT carcinogenic effects were seen in the liver of male and female rats and mice, the nasal cavity of male and female rats, and the lung and forestomach of female mice. In rodents, DMPT is distributed to many of the sites where toxic and carcinogenic effects occurred. DMPT-induced oxidative damage at these target sites may be one mechanism for the treatment-related lesions. Methemoglobinemia, as seen in these DMPT studies, is caused by oxidation of the heme moiety, and this end point served as an early alert for other target organ toxicities and carcinogenic responses that followed with longer term exposure.
Inhalation of diacetyl, a butter flavoring, causes airway responses potentially mediated by sensory nerves. This study examines diacetyl-induced changes in sensory nerves of tracheal epithelium. Rats (n = 6/group) inhaled 0-, 25-, 249-, or 346-ppm diacetyl for 6 hr. Tracheas and vagal ganglia were removed 1-day postexposure and labeled for substance P (SP) or protein gene product 9.5 (PGP9.5). Vagal ganglia neurons projecting to airway epithelium were identified by axonal transport of fluorescent microspheres intratracheally instilled 14 days before diacetyl inhalation. End points were SP and PGP9.5 nerve fiber density (NFD) in tracheal epithelium and SP-positive neurons projecting to the trachea. PGP9.5-immunoreactive NFD decreased in foci with denuded epithelium, suggesting loss of airway sensory innervation. However, in the intact epithelium adjacent to denuded foci, SP-immunoreactive NFD increased from 0.01 ± 0.002 in controls to 0.05 ± 0.01 after exposure to 346-ppm diacetyl. In vagal ganglia, SP-positive airway neurons increased from 3.3 ± 3.0% in controls to 25.5 ± 6.6% after inhaling 346-ppm diacetyl. Thus, diacetyl inhalation increases SP levels in sensory nerves of airway epithelium. Because SP release in airways promotes inflammation and activation of sensory nerves mediates reflexes, neural changes may contribute to flavorings-related lung disease pathogenesis.
Electronic images of histopathological changes are commonly and increasingly used in toxicologic pathology for morphological evaluation, illustration, peer review, or reporting. Toxicity studies in which cell proliferation is an end point are also pivotal in determining the carcinogenic potential of new molecules. In this article, we describe the approach of the European Cell Proliferation and Apoptosis working group (CEPA) for performing cell proliferation studies and morphometry using electronic images. The Society of Toxicologic Pathology (STP) has published a position statement on handling of pathology image data in compliance with 21 Code of Federal Regulations (CFR) Parts 58 and 11. CEPA supports the STP position and shares the issues involved in the use of electronic images in pathology. However, considering the experience and current know-how of members, particularly in conducting cell proliferation studies, CEPA would like to recommend in this article that electronic images acquired using state-of-the-art slide imaging techniques, including whole slide scanning, need not be considered as raw data, and therefore are not subject to 21 CFR Parts 58 and 11 regulations for archiving. In this article, we detail the reasons why we come to this proposal and we describe the measures that are taken to ensure Good Laboratory Practice–compliant execution of cell proliferation studies that include acquisition and validation of imaging and image analysis systems, development and validation of methods for their intended use, formulation, and use of standard operating procedures.
In recent years, there has been considerable activity to identify urinary biomarkers of nephrotoxicity as noninvasive measurements with greater sensitivity and specificity than traditional biomarkers, such as serum creatinine and blood urea nitrogen. Our study aimed to use cisplatin-treated rats to evaluate the use of immunohistochemistry directed at multiple urinary biomarkers in kidney tissue. Tissue levels were compared to urinary levels of these biomarkers to demonstrate tissue specificity and sensitivity. These techniques could also be used in studies where urine samples are not available, such as retrospective studies in drug safety testing, to demonstrate the potential utility of using these biomarkers in future preclinical or clinical studies. All of the biomarkers investigated showed either an increase (kidney injury molecule [KIM-1], osteopontin [OPN], and, clusterin) or a decrease (alpha-glutathione S-transferase and trefoil factor 3) except beta 2 microglobulin (β2MG) that showed no significant changes 5 days after 1.0 mg/kg or 2.5 mg/kg cisplatin treatment. All of the biomarkers except β2MG showed utility as tissue biomarkers, but KIM-1 and OPN expression correlated closely with urinary biomarker measurements and reflect tissue damage. Future studies are needed to determine the wider application of these two markers for detecting renal toxicity following administration of other nephrotoxicants.
Conjugation of therapeutic proteins with high molecular weight polyethylene glycols (HMW PEGs) is used to extend the half-life of biologics. To evaluate the effects of HMW PEGs in animals, we used an immunohistochemical procedure to study the tissue distribution and toxicity of unconjugated HMW PEGs in rats given 100 mg/kg 10KPEG, 20KPEG, or 40KPEG intravenously. Both the PEG cellular distribution and the histology were different between groups. In 10KPEG and 20KPEG groups, PEG immunoreactivity was most prominent in the renal tubule epithelium and in alveolar macrophages and hepatic Kupffer cells and cellular vacuolation was absent. In contrast, rats given 40KPEG had strong PEG immunoreactivity in splenic subcapsular red pulp macrophages, renal interstitial macrophages, and choroid plexus epithelial cells that was frequently associated with cytoplasmic vacuolation. While the vacuolation appeared to be an adaptive response, there was focal renal tubular epithelial degeneration associated with strong PEG immunoreactivity in one rat given 40KPEG. These data indicate that both the tissue distribution and the vacuolation observed with unconjugated HMW PEGs are markedly influenced by the molecular weight of the PEG and that when vacuolation is observed it is likely an adaptive change that is associated with PEG cytoplasmic immunoreactivity.
Ricin toxin, a type 2 ribosome-inactivating protein and a category B bioterrorism agent, is produced from the seeds of castor oil plant (Ricinus communis). Chronic pathological changes in survivors of aerosolized ricin exposure have not been reported in primates. Here we compare and contrast the pathological changes manifested between rhesus macaques (RM) that succumbed to lethal dose of ricin (group I) and survivor RM exposed to low dose of ricin (group II). All animals in group I exhibited severe diffuse, necrotizing bronchiolitis and alveolitis with fibrinopurulent bronchointerstitial pneumonia, massive alveolar, perivascular and peribronchial/bronchiolar edema with hemorrhage, and necropurulent and hemorrhagic tracheobronchial lymphadenitis. All animals from group II had multifocal, fibrosing interstitial pneumonia with prominent alveolar histiocytosis and type II pneumocyte hyperplasia. Subacute changes like infiltration by lymphocytes and plasma cells and persistence of edematous fluid were occasionally present in lung and tracheobronchial lymph nodes. The changes appear to be a continuum wherein the inflammatory response shifts from an acute to subacute/chronic reparative process if the animals can survive the initial insult.
We observed spontaneous dwarfism among Wistar Hannover GALAS rats, caused by primary hypothyroidism. The dwarf (D) rats showed markedly high serum thyroid-stimulating hormone (TSH) values. In this study, we investigated the incidence and histopathological characteristics of the proliferative lesions of the thyroid follicular cells in our D rats. Gross enlargement of thyroid and histological vacuolar change of the thyroid follicular cells with age-related progression suggesting diffuse hypertrophy/hyperplasia were seen in all the D rats. In addition, focal proliferative lesions—namely hyperplasias, activated hyperplasias, adenomas, and adenocarcinomas—were seen in D rats aged over 50 weeks. Taken together, these results suggested that the sustained stimulation of elevated levels of serum TSH caused and enhanced diffuse hypertrophy of thyroid follicular cells and subsequent development of focal proliferative lesions.
3,3',4,4'-tetrachloroazobenzene (TCAB) is a contaminant formed during manufacture of various herbicide compounds. A recent National Toxicology Program study showed B6C3F1 mice exposed to TCAB developed a treatment-related increase in lung carcinomas in the high-dose group, and urethral carcinomas, an extremely rare lesion in rodents, in all dose groups. As the potential for environmental exposure to TCAB is widespread, and the mechanisms of urethral carcinogenesis are unknown, TCAB-induced urethral and pulmonary tumors were evaluated for alterations in critical human cancer genes, Kras and Tp53. Uroplakin III, CK20, and CK7 immunohistochemistry was performed to confirm the urothelial origin of urethral tumors. TCAB-induced urethral carcinomas harbored transforming point mutations in K-ras (38%) and Tp53 (63%), and 71% displayed nuclear TP53 expression, consistent with formation of mutant protein. Transition mutations accounted for 88% of Tp53 mutations in urethral carcinomas, suggesting that TCAB or its metabolites target guanine or cytosine bases and that these mutations are involved in urethral carcinogenesis. Pulmonary carcinomas in TCAB-exposed animals harbored similar rates of Tp53 (55%) and Kras (36%) mutations as urethral carcinomas, suggesting that TCAB may induce mutations at multiple sites by a common mechanism. In conclusion, TCAB is carcinogenic at multiple sites in male and female B6C3F1 mice through mechanisms involving Tp53 and Kras mutation.
Inorganic arsenic (arsenite and arsenate) at high exposures is a known human carcinogen, inducing tumors of the urinary bladder, skin, and lungs. In two experiments, we examined the urothelial proliferative effects of treatment with 173 ppm sodium arsenite (100 ppm arsenic) in the drinking water for 6 and 24 hr, and 3, 7, and 14 days in female F344 rats and 43.3 ppm sodium arsenite (25 ppm arsenic) in female C57BL/6 wild-type and arsenic (+3 oxidation state) methyltransferase knockout (As3mt KO) mice that are unable to methylate arsenicals. In the rat and both mouse genotypes, scanning electron microscopy showed cytotoxic urothelial changes as early as 6 hr after the start of arsenic exposure. The severity of AsIII-induced cytotoxic urothelial changes increased over time in the rat and in the As3mt KO mouse. Light microscopy showed an increase in urothelial hyperplasia in the rat. No significant increases in bromodeoxyuridine-labeling index were observed. The data support the hypothesis that the sequence of events in the mode of action for urothelial effects of orally administered inorganic arsenic in the rat and mouse involves superficial cytotoxicity with consequent regenerative increased cell proliferation similar to the findings associated with the administration of dimethylarsinic acid (DMAV) in rats.
We recently reported results that erythropoiesis-stimulating agent (ESA)–related thrombotic toxicities in preclinical species were not solely dependent on a high hematocrit (HCT) but also associated with increased ESA dose level, dose frequency, and dosing duration. In this article, we conclude that sequelae of an increased magnitude of ESA-stimulated erythropoiesis potentially contributed to thrombosis in the highest ESA dose groups. The results were obtained from two investigative studies we conducted in Sprague-Dawley rats administered a low (no thrombotic toxicities) or high (with thrombotic toxicities) dose level of a hyperglycosylated analog of recombinant human erythropoietin (AMG 114), 3 times weekly for up to 9 days or for 1 month. Despite similarly increased HCT at both dose levels, animals in the high-dose group had an increased magnitude of erythropoiesis measured by spleen weights, splenic erythropoiesis, and circulating reticulocytes. Resulting prothrombotic risk factors identified predominantly or uniquely in the high-dose group were higher numbers of immature reticulocytes and nucleated red blood cells in circulation, severe functional iron deficiency, and increased intravascular destruction of iron-deficient reticulocyte/red blood cells. No thrombotic events were detected in rats dosed up to 9 days suggesting a sustained high HCT is a requisite cofactor for development of ESA-related thrombotic toxicities.
We previously reported an increased incidence of thrombotic toxicities in Sprague-Dawley rats administered the highest dose level of a hyperglycosylated analog of recombinant human erythropoietin (AMG 114) for 1 month as not solely dependent on high hematocrit (HCT). Thereafter, we identified increased erythropoiesis as a prothrombotic risk factor increased in the AMG 114 high-dose group with thrombotic toxicities, compared to a low-dose group with no toxicities but similar HCT. Here, we identified pleiotropic cytokines as prothrombotic factors associated with AMG 114 dose level. Before a high HCT was achieved, rats in the AMG 114 high, but not the low-dose group, had imbalanced hemostasis (increased von Willebrand factor and prothrombin time, decreased antithrombin III) coexistent with cytokines implicated in thrombosis: monocyte chemotactic protein 1 (MCP-1), MCP-3, tissue inhibitor of metalloproteinases 1, macrophage inhibitory protein-2, oncostatin M, T-cell-specific protein, stem cell factor, vascular endothelial growth factor, and interleukin-11. While no unique pathway to erythropoiesis stimulating agent-related thrombosis was identified, cytokines associated with increased erythropoiesis contributed to a prothrombotic intravascular environment in the AMG 114 high-dose group, but not in lower dose groups with a similar high HCT.
We conducted a retrospective analysis of publicly available preclinical toxicology studies with erythropoiesis-stimulating agents (ESAs) to examine common adverse events in rats, Beagle dogs, and cynomolgus monkeys. Mortality and/or thrombotic events were reported sporadically in a subset of studies and attributed to the high hematocrit (HCT) achieved in the animals. However, similarly high HCT was achieved in both high-dose and low-dose groups, but there were no reported adverse events in the low-dose group suggesting HCT was not the sole contributing factor leading to toxicity. Our analysis indicated that increased dose, dose frequency, and dosing duration in addition to high HCT contributed to mortality and thrombosis. To further evaluate this relationship, the incidence of toxicities was compared in rats administered an experimental hyperglycosylated analog of recombinant human erythropoietin (AMG 114) at varying dosing schedules in 1-month toxicity studies. The incidence of mortality and thrombotic events increased in higher dose groups and when dosed more frequently, despite a similarly high HCT in all animals. The results from the investigative study and retrospective analysis demonstrate that ESA-related toxicities in preclinical species are associated with dose level, dose frequency, and dosing duration, and not solely dependent upon a high HCT.
Dexamethasone was given in 2 oral dosing regimens with repeat dose oral administration of the gamma secretase inhibitor (GSI), PF-03084014, in Sprague-Dawley (SD) rats in order to evaluate the effects of coadministration of dexamethasone on GSI-induced goblet cell hyperplasia (GCH) in the intestinal tract. Safety end points were evaluated in 1 week and 1 month studies. The dosing regimens tested in the 1-month studies included a 1-week pretreatment with 1.0 mg/kg dexamethasone followed by a 3-week repeat dose treatment with 100 mg/kg GSI or concurrent intermittent treatment with 1.0 mg/kg dexamethasone on weeks 1 and 3 and repeat dose treatment with 100 mg/kg GSI for 4 weeks. Pretreatment with dexamethasone for 1 week transiently mitigated the severity of intestinal GCH for up to 1 week. Intermittent coadministration of dexamethasone on weeks 1 and 3 with GSI repeat dosing for 4 weeks mitigated intestinal GCH for up to 4 weeks post treatment. Treatment-related morbidity and mortality occurred on day 7 with 150 mg/kg GSI and 5 mg/kg dexamethasone coadministration, and on days 13, 14, and 23 with 100 mg/kg GSI and 1 mg/kg dexamethasone coadministration.
In preclinical studies, it is important to know whether the animals used are sexually mature or not. Precise data have not yet been published, however, about the histological features of the female reproductive organs during the peripubertal period or about the age of acquisition of sexual maturity in the minipig. The histological characteristics of the genital organs of female control minipigs from toxicology studies were described and, based on the presence of ovarian corpora lutea, used to assess the age at which maturity was reached. Only 50% of females can be considered mature at about 6.5 months old (a body weight of 11.8 kg), and 100% were not mature until about 7.5 months old (13.1 kg), although it is said that females reach sexual maturity at the age of approximately 5 months, by the time the body weight is about 10 to 12 kg. The uterine weights of mature females were higher than 94.4 g, whereas the maximum weight reached in the immature females was 55.2 g. In contrast, the differences between immature and mature ovarian weights were not significant. The histological appearance of the mature vagina in the various stages of the estrous cycle is also described.
Drug-induced pancreatitis (DIP) is an underdiagnosed condition that lacks sensitive and specific biomarkers. To better understand the mechanisms of DIP and to identify potential tissue biomarkers, we studied experimental pancreatitis induced in male C57BL/6 mice by intraperitoneal injection of caerulein (10 or 50 μg/kg) at 1-hr intervals for a total of 7 injections. Pancreata from caerulein-treated mice exhibited consistent acinar cell autophagy and apoptosis with infrequent necrosis. Kinetic assays for serum amylase and lipase also showed a dose-dependent increase. Terminal deoxynucleotidyl transferase–mediated biotin-dNTP nick labeling (TUNEL) detected dose-dependent acinar cell apoptosis. By light microscopy, autophagy was characterized by the formation of autophagosomes and autolysosomes (ALs) within the cytoplasm of acinar cells. Immunohistochemical studies with specific antibodies for proteins related to autophagy and pancreatic stress were conducted to evaluate these proteins as potential biomarkers of pancreatitis. Western blots were used to confirm immunohistochemical results using pancreatic lysates from control and treated animals. Autophagy was identified as a contributing process in caerulein-induced pancreatitis and proteins previously associated with autophagy were upregulated following caerulein treatment. Autophagosomes and ALs were found to be a common pathway, in which cathepsins, lysosome-associated membrane protein 2, vacuole membrane protein 1, microtubule-associated protein 1 light chain 3 (LC3), autophagy-related protein 9, Beclin1, and pancreatitis-associated proteins were simultaneously involved in response to caerulein stimulus. Regenerating islet-derived 3 gamma (Reg3), a pancreatic acute response protein, was dose-dependently induced in caerulein-treated mice and colocalized with the autophagosomal marker, LC3. This finding supports Reg3 as a candidate biomarker for pancreatic injury.
Liver resection is a suitable option for the treatment of certain hepatic conditions, particularly hepatocarcinomas, in patients with cirrhosis. However, this disease impairs liver regeneration, which increases the risk of liver failure and postoperative death. Supportive treatments for regeneration of the remaining liver may be useful for the recovery of these patients. We demonstrated that nutritional hepatotrophic factors (NHF) is an effective regenerative stimulus for cirrhotic livers in rats subjected to partial hepatectomy (PH). The rats with thioacetamide-induced cirrhosis were subjected to PH, and they were divided into 2 groups. One group received intraperitoneal administration of NHF, and the other group received saline solution. After 12 days, biometric data, collagen content, hepatocyte regeneration (proliferation cell nuclear antigen immunochemistry), and profibrotic gene expression (Collagen-α1, matrix metalloproteinase 2, tissue inhibitor of metalloproteinase 1, and transforming growth factor beta 1) were assessed. The results indicated that the rats treated with NHF after PH had an increased liver size, a reduced amount of collagen, and a higher hepatocyte proliferation index compared with the rats that underwent PH alone. In addition, collagen-α1 gene expression was decreased in the NHF-treated rats. Thus, postoperative improvement in the liver morphology following NHF treatment may cause a significant decrease in the risk of liver failure and mortality after hepatic resection.
During 2011, International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice (INHAND) Global Editorial Steering Committee representatives had discussions with representatives of the Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER), Clinical Data Interchange Standards Consortium (CDISC), and the National Cancer Institute (NCI) Enterprise Vocabulary Services (EVS) to examine the potential use of INHAND terminology for SEND (Standard for Exchange of Nonclinical Data) submissions to the FDA. The interest in utilizing the INHAND nomenclature, based on input from industry and government toxicologists as well as information technology specialists, suggests that there will be wide acceptance of INHAND nomenclature. The purpose of this article is 2-fold: (1) to provide a brief historical background on the development of SEND and how it is structured and (2) to discuss the impact of SEND on toxicologic pathology and the role of INHAND.
In response to growing concerns that environmental chemicals may have adverse effects on human health by altering the endocrine system, the Endocrine Disruptor Screening Program (EDSP), under the auspices of the United States Environmental Protection Agency (U.S. EPA), recently instituted a Tier I battery of tests including a female pubertal assay. This assay requires dosing of female rats from postnatal day (PND) 22 through PND 42 (or 43), the period of pubertal development in the rat, to identify test articles that may have estrogenic or antiestrogenic effects, or may alter hormones or neurotransmitters. While certain landmarks in female rat reproductive development are published, little is published on the microscopic appearance of the female reproductive tract during prepubertal and pubertal development. In this study, reproductive tissues from three female Sprague-Dawley rats were collected each day from PND 20 through PND 50, such that tissues from a total of 93 rats were collected throughout the prepubertal and pubertal period. Tissues were formalin-fixed, trimmed, paraffin-embedded, sectioned at 5-µm thickness, and examined microscopically. The major histologic features of the female reproductive tract throughout this critical period were described in detail. This information will help pathologists interpret findings observed in female pubertal assays.
A panel of pathologists (Panel) was formed to evaluate the pathogenesis and human relevance of tumors that developed in the fundic region of rat stomachs in carcinogenicity and mechanistic studies with alachlor and butachlor. The Panel evaluated stomach sections stained with hematoxylin and eosin, neuron-specific enolase, and chromogranin A to determine the presence and relative proportion of enterochromaffin-like (ECL) cells in the tumors and concluded all tumors were derived from ECL cells. Biochemical and pathological data demonstrated the tumor formation involved a nongenotoxic threshold mode of action (MOA) initially characterized by profound atrophy of the glandular fundic mucosa that affected gastric glands, but not surface epithelium. This resulted in a substantial loss of parietal cells and a compensatory mucosal cell proliferation. The loss of parietal cells caused a marked increase in gastric pH (hypochlorhydria), leading to sustained and profound hypergastrinemia. The mucosal atrophy, together with the increased gastrin, stimulated cell growth in one or more ECL cell populations, resulting in neoplasia. ECL cell autocrine and paracrine effects led to dedifferentiation of ECL cell tumors. The Panel concluded the tumors develop via a threshold-dependent nongenotoxic MOA, under conditions not relevant to humans.
In most thermally treated products, a series of alkylated furan derivatives have been found, in particular 2-substituted alkylfurans such as 2-methylfuran. These methyl analogs are metabolically activated in a similar fashion as the parent furan, yielding highly reactive unsaturated dialdehydes. There is currently limited toxicological data available for 2-methyl furan exposure by any route that makes conducting a risk assessment difficult. In this pilot study, we report the general toxicology findings affecting tissue morphology, histopathology, clinical biochemistry, and hematology in a 28-day gavage study. The liver was the primary target organ that developed dose-dependent toxicity. Relative liver weights were increased by 42% at 25.0 mg/kg/body weight (bw)/day. Histological changes in the liver were observed at 0.4, 1.5, 3.0, 6.0, 12.0, and 25.0 mg/kg bw/day. These changes were not accompanied by clinical changes in the serum enzyme markers such as alanine transaminase, alkaline phosphatase, and aspartate transaminase. Clinical biochemistry markers for kidney were altered, but these were not accompanied by histological changes. The prostate was significantly decreased in size at the 25.0 mg/kg bw/day dose of 2-methyfuran. Some hematological parameters were also altered.
Establishment of appropriate animal models is an important step in exploring the mechanisms of drug-induced ototoxicity. In the present study, using guinea pigs we compared cochlear lesions induced by cisplatin administered in two regimens: consecutive application alone and in combination with furosemide. The effects of furosemide alone were also evaluated; it was found to cause temporary hearing loss and reversible damage to the stria vascularis. Consecutive application of cisplatin alone appeared to be disadvantageous because it resulted in progressive body weight loss and higher mortality compared to the combined regimen, which used a smaller cisplatin dose. The combined regimen resulted in comparable hearing loss and hair cell loss but a markedly lower mortality. However, their coadministration failed to cause similar damage to spiral ganglion neurons (SGN), as seen in animals that received cisplatin alone. This difference suggests that the combined regimen did not mimic the damage to cochlear neuronal innervation caused by the clinical application of cisplatin. The difference also suggests that the SGN lesion is not caused by cisplatin entering the cochlea via the stria vascularis.
The immune system represents a key defense mechanism against potential pathogens and adverse non-self materials. During pregnancy, the placenta is the point of contact between the maternal organism and non-self proteins of the fetal allograft and hence undoubtedly fulfils immune functions. In the placenta bacteria, foreign (non-self) proteins and proteins that might be introduced in toxicological studies or by medication are barred from reaching the progeny, and the maternal immune system is primed for acceptance of non-maternal fetal protein. Both immunologic protection of the fetus and acceptance of the fetus by the mother require effective mechanisms to prevent an immunologic fetomaternal conflict and to keep both organisms in balance. This is why the placenta requires toxicological consideration in view of its immune organ function. The following articles deal with placenta immune-, control-, and tolerance mechanisms in view of both fetal and maternal aspects. Furthermore, models for experimental access to placental immune function are addressed and the pathological evaluation is elucidated. "The Placenta as an Immune Organ and Its Relevance in Toxicological Studies" was subject of a continuing education course at the 2012 Society of Toxicologic Pathology meeting held in Boston, MA.
This review summarizes the potential and also some limitations of using human placentas, or placental cells and structures for toxicology testing. The placenta contains a wide spectrum of cell types and tissues, such as trophoblast cells, immune cells, fibroblasts, stem cells, endothelial cells, vessels, glands, membranes, and many others. It may be expected that in many cases the relevance of results obtained from human placenta will be higher than those from animal models due to species specificity of metabolism and placental structure. For practical and economical reasons, we propose to apply a battery of sequential experiments for analysis of potential toxicants. This should start with using cell lines, followed by testing placenta tissue explants and isolated placenta cells, and finally by application of single and dual side ex vivo placenta perfusion. With each of these steps, the relative workload increases while the number of feasible repeats decreases. Simultaneously, the predictive power enhances by increasing similarity with in vivo human conditions. Toxic effects may be detected by performing proliferation, vitality and cell death assays, analysis of protein and hormone expression, immunohistochemistry or testing functionality of signaling pathways, gene expression, transport mechanisms, and so on. When toxic effects appear at any step, the subsequent assays may be cancelled. Such a system may be useful to reduce costs and increase specificity in testing questionable toxicants. Nonetheless, it requires further standardization and end point definitions for better comparability of results from different toxicants and to estimate the respective in vivo translatability and predictive value.
Oral gavage studies with β-myrcene in male F344 rats showed a complex renal pathology comprising both alpha2u-globulin (α2u-g) nephropathy, an unusual nephrosis involving the outer stripe of outer medulla (OSOM), and an increased incidence of renal tubule tumors by 2 years. In the 90-day and 2-year studies, respectively, α2u-g nephropathy and linear papillary mineralization were observed in males at the two lower doses but were absent from the high dose. Nephrosis was characterized by dilation of the S3 tubules, nuclear enlargement (including karyomegaly), and luminal pyknotic cells, all in the outermost OSOM. Nephrosis was minimal at the higher doses in the 90-day study, but progressed to a severe grade in males dosed with 1,000 mg/kg for 2 years. Renal tubule tumors developed in treated groups with incidences up to 30% in the 250 and 500 mg/kg male dose groups. Tumors at the lower doses in males may have been associated with α2u-g nephropathy, while those at higher doses in both sexes may have been due to the nephrosis. Because β-myrcene induced a complex spectrum of renal pathology, the α2u-g nephropathy mechanism cannot be the sole mechanism of carcinogenesis in these rats.
This article reviews the regulatory guidelines that provide for the inclusion of recovery groups in toxicology studies, presents the challenges in the design and interpretation of nonclinical recovery studies, and summarizes the best practices for the role of an anatomic pathologist regarding toxicology studies with recovery groups. Evaluating the potential recovery of histopathologic findings induced by a biopharmaceutical requires the active participation of one or more anatomic pathologists. Their expertise is critical in risk assessment regarding the potential for recovery as well as providing scientific guidance in the design and evaluation of studies with recovery groups.
Cardiac troponin (cTn) has been utilized to assess acute myocardial injury, but the cTn response in active/ongoing chronic injury is not well documented. The purpose of this study was to characterize the cardiac troponin I (cTnI), cardiac troponin T (cTnT), high-sensitivity cTnI, hematology, and clinical chemistry responses in rats treated with doxorubicin. Rats treated with 1, 2, or 3 mg/kg/week (wk) of doxorubicin for 2, 4, or 6 wks were sacrificed after 0, 2, or 4 wks of recovery and compared to untreated controls and animals treated with doxorubicin/dexrazoxane (50 mg/kg/wk) or etoposide (1 and 3 mg/kg/wk). The incidence and mean magnitude of cTn response increased with increasing dose and/or duration of doxorubicin treatment. Conversely, dexrazoxane/doxorubicin was partially protective for cardiotoxicity, and minimal cardiotoxicity occurred with etoposide treatment. Both cTnI and cTnT effectively identified doxorubicin-induced injury as indicated by vacuolation of cardiomyocytes of the atria/ventricles. The association between the cTn responses and histological changes was greater at the higher total exposures, but the magnitude of cTn response did not match closely with histologic grade. The high-sensitivity cTnI assay was also effective in identifying cardiac injury. Alterations occurred in the hematology and clinical chemistry parameters and reflected both dose and duration of doxorubicin treatment.
The constitutive androstane receptor (CAR) is essential for Cyp2b induction, liver hypertrophy, and hepatocarcinogenesis in response to phenobarbital (PB). Liver hypertrophy with Cyp2b induction is a major mode of action of hepatocarcinogenesis in rodents. However, it remains unclear whether CAR is involved in the response to many other nongenotoxic hepatocarcinogens besides PB. In this study, we investigated CAR involvement in liver hypertrophy and hepatocarcinogenesis of Cyp2b-inducing nongenotoxic hepatocarcinogens, piperonyl butoxide (PBO), and decabromodiphenyl ether (DBDE), using wild-type and CAR knockout (CARKO) male mice. PB was used as the positive control. In the wild-type mice, 4-week treatment with PBO, DBDE, or PB induced hepatocellular hypertrophy with increased Cyp2b10 messenger RNA and Cyp2b protein expression. In CARKO mice, only PBO showed liver hypertrophy with Cyp2b10 and Cyp3a11 induction. After 27-week treatment following diethylnitrosamine initiation, PBO and PB generated many eosinophilic altered foci/adenomas in wild-type mice; however, the lesions were far less frequent in CARKO mice. DBDE increased the multiplicity of basophilic altered foci/adenomas in wild-type and CARKO mice. Our findings indicate that murine CAR plays major roles in hepatocarcinogenesis but not in liver hypertrophy of PBO. DBDE may act via CAR-independent pathways during hepatocarcinogenesis.
This short review is derived from the peer-reviewed literature and the experience and case materials of the authors. Brief illustrated summaries are presented on the gross and histologic normal anatomy of rodent and macaque placentas, including typical organ weights, with comments on differences from the human placenta. Common incidental findings, background lesions, and induced toxic lesions are addressed, and a recommended strategy for pathologic evaluation of placentas is provided.
During pregnancy, the maternal immune system is challenged by the semiallogeneic fetus, which must be tolerated without compromising fetal or maternal health. This review updates the systemic and local immune changes taking place during human pregnancy, including some examples in rodents. Systemic changes are induced by contact of maternal blood with placental factors and include enhanced innate immunity with increased activation of granulocytes and nonclassical monocytes. Although a bias toward T helper (Th2) and regulatory T cell (Treg) immunity has been associated with healthy pregnancy, the relationship between different circulating Th cell subsets is not straightforward. Instead, these adaptations appear most evidently at the fetal–maternal interface, where for instance Tregs are enriched and promote fetal tolerance. Also innate immune cells, that is, natural killer cells and macrophages, are enriched, constituting the majority of decidual leukocytes. These cells not only contribute to immune regulation but also aid in establishing the placenta by promoting trophoblast recruitment and angiogenesis. Thus, proper interaction between leukocytes and placental trophoblasts is necessary for normal placentation and immune adaptation. Consequently, spontaneous maladaptation or interference of the immune system with toxic substances may be important contributing factors for the development of pregnancy complications such as preeclampsia, preterm labor, and recurrent miscarriages.
Clinically, girls appear to be more sensitive than boys to the cardiotoxic effects of doxorubicin, whereas the opposite may be true for adults. To identify and characterize potential sex-related differences, adult male and female spontaneously hypertensive rats (SHR; some ovariectomized [OVX]) received 1 mg/kg of doxorubicin or saline iv weekly for 9, 10, or 12 weeks. Weight gain was slower in treated males. Serum concentrations of cholesterol and triglycerides increased and those of albumin decreased in both sexes, but changes were more pronounced in treated males. Treated males had significantly more severe cardiomyopathy scores and higher serum levels of cTnT than females. The increased cardiotoxicity was accompanied by higher numbers of cardiac mast cells (MCs) and percentage of cardiac MCs undergoing degranulation. Doxorubicin-treated OVX animals had significantly increased numbers of cardiac MCs, more severe myocardial lesions, and elevated serum concentrations of cTnT compared to doxorubicin-treated normal female SHR. The severity of cardiac lesions in the OVX female was similar to that observed in doxorubicin-treated males. This study demonstrated the presence of sex-related differences in the cardiotoxic effects elicited by doxorubicin and identified variations in the level of cardiac MC activity as a factor which could possibly contribute to the male-female dissimilarity.
A factor limiting widespread use of the transgenic rasH2 mouse model for carcinogenicity testing of pharmaceuticals is the paucity of published data on actual drug candidates in rasH2 mice. This report addresses this gap by highlighting rasH2 mouse study data for 10 pharmaceutical candidates. These results were compared with findings in the 2-year studies in Sprague-Dawley rats for the same 10 compounds. In the 6-month rasH2 studies, only 2 of the 10 compounds tested positive for carcinogenicity and these correlated with positive findings in the companion 2-year rat studies. One compound, sunitinib, produced gastroduodenal carcinoma in both sexes and increased hemangiosarcoma in spleen and uterus in female rasH2 mice; in rats it produced gastroduodenal carcinoma and increased pheochromocytoma (males only). The second compound, bazedoxifene, produced ovarian granulosa cell neoplasms in rasH2 mice and rats, and renal tubular neoplasms associated with increased chronic progressive nephropathy only in rats. The higher percentage of carcinogenicity positive rat bioassays could be attributed to rat-specific phenomena with little or low relevance to man. Thus, this article confirms previous reports that rasH2 mice develop rodent-specific neoplasms less frequently than rats and positive findings, when present, are accompanied by similar positive results in the rat.
Cardiovascular safety signals in nonclinical studies remain among the main reasons for drug attrition during pharmaceutical research and development. Drug-induced changes can be functional and/or associated with morphological alterations in the normal heart histology. It is therefore crucial to understand the normal variations in histology to discriminate test article–related changes from background lesions. Rodent progressive cardiomyopathy is probably the most commonly encountered change in control animals of nonclinical toxicity studies. A multisite study mimicking standard short-term toxicity studies using young male Sprague-Dawley rats was performed to better characterize this finding. Using an enhanced sectioning method for this research study, it was observed that the incidence of background cardiomyopathy was 100%. The vast majority of the microscopic findings were inflammatory in nature, with associated necrotic changes (defined as necrosis/inflammatory cell infiltrate) and these changes were mainly located in the myocardium of the mid region of the ventricles (the left side being predominantly affected). The monitored environmental factors in this study (multiple facilities, study duration, handling) did not have an effect on the incidence or severity of the spontaneous cardiomyopathy. In addition, cardiac-specific serum troponin levels were measured and were within the published control range.
The Society of Toxicologic Pathology charged a Nervous System Sampling Working Group with devising recommended practices to routinely screen the central nervous system (CNS) and peripheral nervous system (PNS) in Good Laboratory Practice–type nonclinical general toxicity studies. Brains should be weighed and trimmed similarly for all animals in a study. Certain structures should be sampled regularly: caudate/putamen, cerebellum, cerebral cortex, choroid plexus, eye (with optic nerve), hippocampus, hypothalamus, medulla oblongata, midbrain, nerve, olfactory bulb (rodents only), pons, spinal cord, and thalamus. Brain regions may be sampled bilaterally in rodents using 6 to 7 coronal sections, and unilaterally in nonrodents with 6 to 7 coronal hemisections. Spinal cord and nerves should be examined in transverse and longitudinal (or oblique) orientations. Most Working Group members considered immersion fixation in formalin (for CNS or PNS) or a solution containing acetic acid (for eye), paraffin embedding, and initial evaluation limited to hematoxylin and eosin (H&E)-stained sections to be acceptable for routine microscopic evaluation during general toxicity studies; other neurohistological methods may be undertaken if needed to better characterize H&E findings. Initial microscopic analyses should be qualitative and done with foreknowledge of treatments and doses (i.e., "unblinded"). The pathology report should clearly communicate structures that were assessed and methodological details. Since neuropathologic assessment is only one aspect of general toxicity studies, institutions should retain flexibility in customizing their sampling, processing, analytical, and reporting procedures as long as major neural targets are evaluated systematically.
Cynomolgus monkeys dosed with a therapeutic monoclonal antibody (mAbY.1) at ≥50 mg/kg had unexpected acute thrombocytopenia (nadir ~3,000 platelets/µl), sometimes with decreases in red cell mass. Increased activated macrophages, mitotic figures, and erythrophagocytosis were observed in the spleen. Binding of mAbY.1 to cynomolgus peripheral blood cells could not be detected in vitro. mAbY.1 induced phagocytosis of platelets by peripheral blood monocytes from cynomolgus monkeys, but not from humans. mAbs sharing the same constant domain (Fc) sequences, but differing from mAbY.1 in their variable domains, bound competitively to and had similar biological activity against the intended target. None of these antibodies had hematologic liabilities in vitro or in vivo. Neither the F(ab’)2 portion of mAbY.1 nor the F(ab’)2 portion on an aglycosylated Fc (IgG1) framework caused phagocytosis of platelets in vitro. These data suggest that the hematologic effects of mAbY.1 in cynomolgus monkeys likely occurred through an off-target mechanism, shown to be driven by 1 to 3 amino acid differences in the light chain. The hematologic effects made mAbY.1 an unsuitable candidate for further development as a therapeutic agent. This example demonstrates that nonclinical safety studies may be essential for understanding off-target effects of mAbs prior to clinical trials.
Spontaneous progressive glomerulonephropathy often occurs in common marmosets. However, there are few detailed reports concerning the age-related progressive process of glomerular changes. We discuss the glomerular changes in the early stage and the progressive changes in the advanced stage of nephropathy. We investigated the kidneys of common marmosets (2–11 years old; 9 males and 12 females) using hematoxylin and eosin, periodic acid–Schiff, periodic acid–methenamine-silver, and Masson’s trichrome (MT) stains and a transmission electron microscope. There was no remarkable change in urine cytology, hematology, or blood chemistry. In the early stage of nephropathy, effacement of podocyte foot processes was observed ultrastructurally even though there were no marked glomerular lesions in the light microscopy. Subsequently, mesangial proliferation occurred from the hilar to peripheral side along the tuft. In the middle stage, red deposits were visible at the glomerular basement membrane (GBM) and the mesangial region directly under the GBM (paramesangial area) with the MT stain. Electron dense deposits were seen at the same area. In the advanced stage, the irregularity became prominent with or without dense deposits. It is necessary to investigate in detail whether the change of podocyte in the early stage was immuno-mediated or due to podocyte failure.
Since 2003, the Tg.rasH2 model has been accepted by regulatory agencies worldwide for 26-week short-term carcinogenicity assays as an alternative to the standard 2-year assays in conventional mice. However, over the decade, the number of actual studies conducted with alternative mouse models has remained low. The primary cause for low acceptance of this model has been lack of a historical database for the incidence of spontaneous lesions. Recently, we published the historical control database on spontaneous tumors in the Tg.rasH2 mice. The purpose of this publication is to present a large database pertaining to the non-neoplastic spontaneous lesions noted in Tg.rasH2 mice from studies conducted at our facility. Lesions that are considered unique in Tg.rasH2 mice are skeletal muscle myopathy, vascular anomalies involving various organs, and mesenteric arterial thrombosis. Other notable lesions are extramedullary hematopoiesis of spleen, subacute inflammatory foci in the liver, and infiltration of histiocytes in the lungs.
Nonhuman primates, particularly rhesus macaques (Macaca mulatta), provide important model systems for studying human reproductive infectious diseases such as human immunodeficiency virus, human papillomavirus, and Chlamydia spp. An understanding of the spectrum of spontaneous cervical disease provides essential context for interpreting experimental disease outcomes in the female reproductive tract. This retrospective study characterizes the incidence of inflammatory and/or proliferative cervicovaginal lesions seen over a 14-year period in a multispecies nonhuman primate colony, focusing on rhesus macaques. The most common observations included a spectrum of lymphocytic accumulation from within normal limits to lymphoplasmacytic cervicitis, and suppurative inflammation with occasional squamous metaplasia or polyp formation. These inflammatory spectra frequently occurred in the context of immunosuppression following experimental simian immunodeficiency virus (SIV) infection. Cervical neoplasias were uncommon and included leiomyomas and carcinomas. Cervical sections from 13 representative cases, with an emphasis on proliferative and dysplastic lesions, were surveyed for leukocyte infiltration, abnormal epithelial proliferation, and the presence of papillomavirus antigens. Proliferative lesions showed sporadic evidence of spontaneous papillomavirus infection and variable immune cell responses. These results underscore the importance of pre screening potential experimental animals for the presence of preexisting reproductive tract disease, and the consideration of normal variability within cycling reproductive tracts in interpretation of cervical lesions.
Acyl-coenzyme A: cholesterol O-Acyltransferase (ACAT) and Acyl-coenzyme A: diacylglycerol O-acyltransferase (DGAT) enzymes play important roles in synthesizing neutral lipids, and inhibitors of these enzymes have been investigated as potential treatments for diabetes and other metabolic diseases. Administration of a Acyl-coenzyme A: diacylglycerol O-acyltransferase 1 (DGAT1) inhibitor with very limited cellular selectivity over ACAT resulted in significant adrenocortical degenerative changes in dogs. These changes included macrosteatotic vacuolation associated with adrenocyte cell death in the zonae glomerulosa and fasciculata and minimal to substantial mixed inflammatory cell infiltration and were similar to those described previously for some ACAT inhibitors in dogs. In the mouse, similar but only transient adrenocortical degenerative changes were seen as well as a distinctive nondegenerative reduction in cortical fine vacuolation. In the marmoset, only the distinctive nondegenerative reduction in cortical fine vacuolation was observed, suggesting that the dog, followed by the mouse, is the most sensitive species for cortical degeneration. Biochemical analysis of adrenal cholesterol and cholesteryl ester indicated that the distinctive reduction in cortical fine vacuolation correlated with a significant reduction in cholesteryl ester in the mouse and marmoset, whereas no significant reduction in cholestryl ester, but an increase in free cholesterol was observed in dogs. Administration of a DGAT1 inhibitor with markedly improved selectivity over ACAT to the marmoset and the mouse resulted in no adrenal pathology at exposures sufficient to cause substantial DGAT1 but not ACAT inhibition, thereby implicating ACAT rather than DGAT1 inhibition as the probable cause of the observed adrenal changes. Recognizing that the distinctive nondegenerative reduction in cortical fine vacuolation in the mouse could be used as a histopathological biomarker for an in vivo model of the more severe changes observed in dogs, the mouse has subsequently been used as a model to select DGAT1 inhibitors free of adrenocortical toxicity.
In this study, male F344 rats were orally exposed to a single dose of aflatoxin B1 (AFB1) at 0, 50, 250, or 1,000 µg/kg body weight (BW) or repeated dose of 0, 5, 10, 25, or 75 µg/kg BW for up to 5 weeks. Biochemical and histological changes were assessed together with the formation of AFB1-lysine adduct (AFB-Lys) and liver foci positive for placental form glutathione S transferase (GST-P+). In single-dose protocol, serum aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) showed dose-related elevation, with maximal changes observed (>100-fold) at day 3 after treatment. Animals that received 250 µg/kg AFB1 showed concurrent bile duct proliferation, necrosis, and GST-P+ hepatocytes at 3 day, followed by liver GST-P+ foci appearance at 1 week. In repeated-dose protocol, bile duct proliferation and liver GST-P+ foci co-occurred after 3-week exposure to 75 µg/kg AFB1, followed by proliferation foci formation after 4 week and dramatic ALT, AST, and CK elevations after 5 weeks. Liver GST-P+ foci were induced temporally and in a dose-related manner. Serum AFB-Lys increased temporally at low doses (5–25 µg/kg), and reached the maximum after 2-week exposure at 75 µg/kg. This integrative study demonstrated that liver GST-P+ cells and foci are sensitive biomarkers for AFB1 toxic effect and correlated with bile duct proliferation and biochemical alterations in F344 rats.
When 100 mg/kg/day of di(n-butyl) phthalate (DBP) was intragastrically administered to pregnant Sprague-Dawley rats throughout gestation days 12 to 21, the male pups had similar body weights with no apparent physical differences (e.g., litter size, sex ratio) compared to that of the vehicle group. However, prominent age-related morphological alterations in the smooth endoplasmic reticulum (sER) of testicular Leydig cells (LCs) were observed once these animals reached puberty. At weeks 5 to 7, the abundant sER with non-dilated cisternae was distributed in LCs. Subsequently, although the number of LCs significantly increased, the amount of sER was significantly decreased at 9 to 14 weeks of age and had disappeared at 17 weeks. In contrast, the number of LCs and the amount of sER in LCs of the lower dose groups (10, 30, and 50 mg/kg/day) were similar to those of the vehicle group. Further, serum testosterone levels in the 100 mg/kg dose group were significantly lower during 5 to 17 weeks of age. While their luteinizing hormone (LH) level was significantly lower at 5 to 7 weeks of age, it became significantly higher during 9 to 17 weeks. The amount of sER in LCs decreased with age with the increase in LCs proliferation and serum LH levels in rat exposed in utero to DBP in a dose-dependent manner.
Bazedoxifene acetate (BZA) is a selective estrogen receptor modulator that is approved in a number of countries for the prevention and/or treatment of osteoporosis in postmenopausal women. To assess carcinogenic potential, BZA was administered ad libitum in the diet to male and female rats for 2 years. The achieved mean dosages of BZA were approximately 1.31 to 56.9 mg/kg/day at dietary concentrations of 0.003% to 0.1%. BZA treatment resulted in a reduction and a delayed onset in total tumor burden in both male and female rats. Survival rates were enhanced due to decreased pituitary and mammary tumors and decreased body weight gain in BZA-treated animals compared with controls. In male rats only, an increase in renal tubular tumors was observed. The greater increase in tumor incidence in male rats given BZA was associated with the increased survival and increased time for development of late onset tumors. These findings are consistent with a non-genotoxic mechanism, unique to male rats, that involves test article–induced corticomedullary mineralization, renal tubular injury, and exacerbation of naturally occurring chronic progressive nephropathy in aged male rats that led to a sequela of proliferative changes and tumor formation.
Image analysis is now routinely employed as a tool in toxicologic pathology to help quantitate end points of efficacy and safety. It is regarded as a proficient and a sensitive technique to generate numerical data that can be easily interrogated for statistical evaluation. Traditional semiquantitative pathology scoring on the other hand is sometimes regarded as less accurate due to the limitations of the scoring systems employed and the day-to-day variations often noted between pathologists. We therefore decided to generate an optimized histochemical staining and image analysis protocol to compare the accuracy of semiquantitative scoring with computerized image analysis. In order to achieve this, we describe a standardized protocol for staining and image analysis that eliminates or minimizes as many sources of error as possible. The results of this experiment demonstrate that despite consistent variations in scoring between two independent pathologists, correlation with image analysis data of 0.91 to 0.95 (Spearman’s Rho test) was achieved. These data indicate that either image analysis or traditional semiquantitative scoring can generate accurate data. As a result of this, it appears that it is equally safe to employ either method dependent upon the complexity and the practicality of the task at hand provided that the experimental conditions are rigorously optimized and rigidly adhered to.
An annular, reddened lesion with mild serosal hemorrhage and no tumorous mass formation was detected in the right uterine horn of a 37-week-old female Sprague-Dawley rat that had postpubertal vaginal bleeding. Histological examination revealed prominent proliferation of the endometrium, which occupied the uterine lumen. There were numerous aberrant vascular spaces filled with erythrocytes, proliferation of stromal cells, and inflammatory infiltrates including hemosiderin-laden macrophages in the endometrium. These vasculatures extended into the myometrium, and in a transverse section of the lesion, they were mostly distributed throughout the circumference of the uterus. They were irregular in shape and interconnected, forming a large vascular sinus and anastomosing reticular channels. In the area with serosal hemorrhage, the muscular layer covering the large irregular vascular space had undergone degeneration and necrosis. The lining cells of the vasculatures were often plump, and they protruded into the lumen and were arranged in a tombstone or hobnail manner. Immunostaining revealed that these cells were positive for von Willebrand factor and CD34. The aberrant vasculatures were not accompanied by pericytes or muscular layer, although a discontinuous muscular wall was present around some of them. From these results, the uterine lesion was diagnosed as a vascular hamartoma.