Is It Adverse, Nonadverse, Adaptive, or Artifact?

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.

Scientific and Regulatory Policy Committee: Recommended ("Best") Practices for Determining, Communicating, and Using Adverse Effect Data from Nonclinical Studies.

Recommendations (best practices) are provided by the Society of Toxicologic Pathology's Adversity Working Group for making consistent interpretations of test article-related effects as "adverse" and assigning a "no observed adverse effect level" (NOAEL) in nonclinical toxicity studies. Adverse is a term indicating "harm" to the test animal, while nonadverse indicates lack of harm. Adverse findings in the study reports should be defined in relation to effects on the test species used and within the context of the given study. Test article-related effects should be described on their own merits, and decisions to consider them as adverse or nonadverse should be justified. Related effects may be discussed together; in particular, markers of toxicity that are not in and of themselves adverse ideally should be discussed in conjunction with the causal toxicity to determine adversity. Adverse findings should be identified in subreports (clinical data, pathology data, etc.) if sufficient information is available, and/or in the final study report as individual or grouped findings, but study NOAELs should be established at the level of the overall study report. Interpretations such as "not biologically relevant" or "not toxicologically important" should be avoided unless defined and supported by scientific rationale. Decisions defining adverse findings and the NOAEL in final study reports should combine the expertise of all contributing scientific disciplines. Where possible, use of NOAELs in data tables should be linked to explanatory text that places them in context. Ideally, in nonclinical summary documents, NOAELs from multiple studies are considered together in defining the most important adverse responses in the most sensitive species. These responses are then considered along with an understanding of their likely mechanisms, as well as other information such as variability in species sensitivity, comparative pathology, reversibility and progression, kinetics, and metabolism of the test substance to help assess human risk.

Histiocytic typhlocolitis in two colony Beagle dogs.

Two young female Beagle dogs in a laboratory colony with clinical signs of loose stools and fecal blood were confirmed to have histiocytic ulcerative colitis by histologic evaluation. This syndrome is well recognized in other dog breeds such as Boxers and related French Bulldogs, Mastiffs, Alaskan malamutes and Doberman Pinschers. Formalin-fixed paraffin sections of large intestine from one dog demonstrated the presence of Escherichia coli strain LF82 by immunohistochemistry and 16S ribosomal RNA gene sequencing. E coli strain LF82 has been implicated in the pathogenesis of Crohn's disease and similar bacteria have been cultured from cases of histiocytic ulcerative colitis in Boxer dogs. Spontaneous histiocytic ulcerative colitis must be differentiated from test article-related findings in nonclinical toxicity studies in Beagle dogs.

The development of subcutaneous sarcomas in rodents exposed to peroxisome proliferators agonists: hypothetical mechanisms of action and de-risking attitude.

Peroxisome proliferator-activated receptors (PPARs) represent therapeutic targets for the management of type 2 diabetes mellitus and dyslipidemia. Rodent carcinogenicity studies have revealed a link between γ and dual γ/α PPAR agonist treatment and the increased incidence of subcutaneous (SC) liposarcomas/fibrosarcomas or hemangiosarcomas, but very little has been reported for potent and selective PPARα agonists. We present a mode of action framework for the development of SC mesenchymal tumors in rodents given PPAR agonists. (1) Tumor promotion results from pharmacologically mediated recruitment (proliferation and differentiation), thermogenesis and adipogenesis of stromovascular cells, and subsequent generation of oxidative free radicals. (2) Tumor initiation consists of chemotype-driven mitochondrial dysfunction causing uncontrolled oxidative stress and permanent DNA damage. Promotion is characterized by enhanced adipogenesis in the SC adipose tissue, where the baseline PPARγ expression and responsiveness to PPARγ ligands is the highest, and by thermogenesis through expression of the uncoupling protein 1 (UCP-1) and the PPARγ co-activator 1 α (PGC-1α), two factors more highly expressed in brown versus white adipose tissue. Initiation is supported by the demonstration of mitochondrial uncoupling and OXPHOS Complexes dysfunction (Complexes III, IV and V) by compounds associated with increased incidences of sarcomas (muraglitazar and troglitazone), but not others lacking malignant tumor effects (pioglitazone, rosiglitazone).

The PPARα agonists fenofibrate and CP-778875 cause increased ß-oxidation, leading to oxidative injury in skeletal and cardiac muscle in the rat.

Weak peroxisome proliferator-activated receptor (PPAR) α agonists (fibrates) are used to treat dyslipidemia. This study compared the effects of the potent and selective PPARα agonist CP-778875 on peroxisomal ß-oxidation and cardiac and/or skeletal muscle injury with those of the weak PPARα agonist fenofibrate. We hypothesized that these muscle effects are mediated through the PPARα receptor, leading to increased ß-oxidation and consequent oxidative stress. CP-778875 (5 or 500 mg/kg) and fenofibrate (600 or 2,000→1,200 mg/kg, dose lowered because of intolerance) were administered to rats for six weeks. Standard end points, serum troponin I, heart and skeletal muscle ß-oxidation of palmitoyl-CoA, and acyl co-oxidase (AOX) mRNA were assessed. Both compounds dose-dependently increased the incidence and/or severity of cardiomyocyte degeneration and necrosis, heart weight, troponin I, and skeletal muscle degeneration. Mean heart ß-oxidation (3.4- to 5.1-fold control) and AOX mRNA (2.4- to 3.2-fold control) were increased with CP-778875 500 mg/kg and both doses of fenofibrate. ß-Oxidation of skeletal muscle was not affected by either compound; however, a significant increase in AOX mRNA (1.6- to 2.1-fold control) was observed with CP-778875 500 mg/kg and both doses of fenofibrate. Taken together, these findings were consistent with PPARα agonism and support the link between increased cardiac and skeletal muscle ß-oxidation and resultant muscle injury in the rat.

Neurotoxic effects of zoniporide: a selective inhibitor of the NA+/H+ exchanger isoform 1.

Zoniporide, an inhibitor of the Na+-H+ exchanger-1, was administered by continuous intravenous infusion to rats and dogs for up to 1 month. In 1-month studies, histological and functional changes were observed in select portions of the peripheral nervous system; however, these findings were not detected in 2-week studies at similar or higher doses. In the 1-month rat study, there was dose-dependent, minimal, focal, or multifocal nerve fiber (axonal) degeneration in the spinal cord and/or sciatic nerve. In a follow-up rat study, findings included slowing of caudal nerve conduction velocity and axonal degeneration in the spinal cord (dorsal funiculus), dorsal roots, dorsal root ganglia (DRG), radial, sciatic, and tibial nerves. In the 1-month dog study, there was impairment of the patellar reflex and associated postural reaction changes, minimal to marked proximal nerve fiber degeneration in the DRG, and minimal nerve fiber degeneration in the dorsal roots and funiculi of the spinal cord. Minimal nerve fiber degeneration of equivocal significance was noted in various peripheral nerves. Taken together, these findings were consistent with a specific effect on peripheral sensory nerve fibers. These studies demonstrated that zoniporide produces clinical, electrophysiologic, and microscopic evidence of peripheral sensory axonopathy and establishes the importance of careful preclinical evaluation of neurological function.

Spontaneous and age-related testicular findings in beagle dogs.

This study was conducted to characterize spontaneous testicular and epididymal microscopic findings in eighty control beagle dogs from toxicity studies. Hypospermatogenesis, characterized by randomly scattered missing spermatids and/or spermatocytes within seminiferous tubules, was observed in 75% of dogs six to seven months of age and declined to fewer than 10% in dogs over eleven months of age. Atrophy/hypoplasia of seminiferous tubules, characterized by subcapsular triangular clusters of tubules containing no germ cells, was observed in 25 to 40% of dogs under twelve months old, decreasing with age to 14 to 17% in dogs twelve to thirty-six months old. Retained spermatids, multinucleate giant cells, intracytoplasmic vacuoles (presumably in Sertoli cells), and swollen spermatocytes were common findings of minimal severity. Six- and seven-month-old dogs had lower testicular weights, less filling of the epididymal tails with sperm, and a two-fold higher incidence of abnormal epididymal content compared to dogs more than eight months of age. Most male beagles were histologically sexually mature by eight to nine months of age. This study confirms published reports that dogs at least ten months of age at necropsy usually are adequate for routine microscopic evaluation of the testes. If evaluation of spermatogenesis is critical, the incidental findings can be minimized by using males over twelve months of age.

A critical role for peroxisomal proliferator-activated receptor-alpha nuclear receptors in the development of cardiomyocyte degeneration and necrosis.

Peroxisomal proliferator-activated receptor (PPAR)-alpha is a ligand-activated transcriptional factor that regulates genes involved in lipid metabolism and energy homeostasis. PPAR-alpha activators, including fibrates, have been used to treat dyslipidemia for several decades. In contrast to their known effects on lipids, the pharmacological consequences of PPAR-alpha activation on cardiac metabolism and function are not well understood. Therefore, we evaluated the role that PPAR-alpha receptors play in the heart. Our studies demonstrate that activation of PPAR-alpha receptors using a selective PPAR-alpha ligand results in cardiomyocyte necrosis in mice. Studies in PPAR-alpha-deficient mice demonstrated that cardiomyocyte necrosis is a consequence of the activation of PPAR-alpha receptors. Cardiac fatty acyl-CoA oxidase mRNA levels increased at doses in which cardiac damage was observed and temporally preceded cardiomyocyte degeneration, suggesting that peroxisomal beta-oxidation correlates with the appearance of microscopic injury and cardiac injury biomarkers. Increased myocardial oxidative stress was evident in mice treated with the PPAR-alpha agonists coinciding with increased peroxisomal biomarkers of fatty acid oxidation. These findings suggest that activation of PPAR-alpha leads to increased cardiac fatty acid oxidation and subsequent accumulation of oxidative stress intermediates resulting in cardiomyocyte necrosis.

Using laser scanning cytometry to measure PPAR-mediated peroxisome proliferation and beta oxidation.

Laser scanning cytometry (LSC) is a new technology that combines the properties and advantages of flow cytometry (FC) and immunohistochemistry (IHC), thus providing qualitative and quantitative information on protein expression with the additional perspective provided by cell and tissue localization. Formalin-fixed, paraffin embedded liver sections from rats exposed to a Peroxisome Proliferator Activated Receptor (PPAR) agonist were stained with antibodies against peroxisomal targeting signal-1 (PTS-1) (a highly conserved tripeptide contained within all peroxisomal enzymes), Acyl CoA oxidase (AOX) (the rate limiting enzyme of peroxisomal beta oxidation), and catalase (an inducible peroxisomal antioxidant enzyme) to evaluate peroxisomal beta oxidation, oxidative stress, and peroxisome proliferation. The LSC showed increased AOX, catalase, and PTS-1 expression in centrilobular hepatocytes that correlated favorably with the microscopic observation of centrilobular hepatocellular hypertrophy and with the palmitoyl CoA biochemical assay for peroxisomal beta oxidation, and provided additional morphologic information about peroxisome proliferation and tissue patterns of activation. Therefore, the LSC provides qualitative and quantitative evaluation of peroxisome activity with similar sensitivity but higher throughput than the traditional biochemical methods. The additional benefits of the LSC include the direct correlation between histopathologic observations and peroxisomal alterations and the potential utilization of archived formalin-fixed tissues from a variety of organs and species.

Immune factors influencing the course of infection with Neospora caninum in the murine host.

This paper investigates the role of specific immune factors on the course of infection in genetic knockout (gko) mice infected with 3 different strains of Neospora caninum. Survival time and parasite persistence were examined in interferon-gamma (IFN-gamma), tumor necrosis factor receptor-2 (TNFR2), interleukin 10 (IL-10), beta 2 microglobulin (beta2M), and inducible nitric oxide synthase (iNOS2) gko or wild-type (wt) mice following infection with either pathogenic (NC-1 or NC-2) or attenuated (NCts-8) N. caninum strains. Infection with NC-1 was 100% lethal in IFN-gamma gko mice, as evidenced by mean survival times of 10-13 days, depending on the challenge dose used. TNFR2 and beta2M gko mice infected with NC-1 or NC-2 strain demonstrated partial susceptibility to disease, as evidenced by histopathology and survival curves. TNFR2 or beta2M gko mice were not susceptible to infection with NCts-8, on the basis of absence of pathology and lack of mortality. Lack of mortality and minimal histopathology scores demonstrated that NC-1, NC-2, and NCts-8 infections were avirulent in IL-10 and iNOS2 gko mice. Adoptive transfer of immune cells from NCts-8 vaccinated normal syngeneic mice into IFN-gamma gko mice significantly (P < 0.05) prolonged mean survival times at all 3 challenge doses of NC-1 but failed to protect against mortality. Interestingly, there was a notable change in the tissue tropism of tachyzoites from the lung and brain in immunocompetent wt, TNFR2 gko, IL-10 gko, beta2M gko, and iNOS2 gko mice to the liver and spleen in IFN-gamma gko mice when challenged with N. caninum. On the basis of these results in gko mice, IFN-gamma is a critical cytokine in the host response against acute neosporosis.

Cystic degeneration/Spongiosis hepatis in rats.

Cystic degeneration/spongiosis hepatis in rats has been proposed to be a preneoplastic and/or neoplastic lesion by some authors, because of its proliferative properties and persistent increased cell turnover rate in stop experiments using hepatocarcinogens, and the assumption that it can develop into a sarcoma. The neoplastic potential of cystic degeneration is questioned in this review article. Cystic degeneration, which appears to derive from altered Ito cells, does not have neoplastic histomorphologic characteristics, although it may be composed of cells with an increased mitotic index. In this regard, persistent proliferation is also seen with other nonneoplastic lesions. Arguments are presented to show that the induced, probably extremely rare sarcoma that was associated with cystic degeneration most likely derives from the very rare induced spherical Ito-cell aggregate with an unusually high cellular turnover rate in rats treated with hepatocarcinogens, and not from cystic degeneration. Also, in none of 12 referenced standard oncogenicity studies with chemically induced cystic degeneration was the lesion associated with mesenchymal (Ito-cell) tumors. Consequently, evidence is lacking that cystic degeneration in rats should be classified as a preneoplastic or neoplastic lesion. The 12 oncogenicity studies in rats with induced cystic degeneration showed a marked sex predilection, with males more likely to develop either spontaneous or chemically induced lesions. In these 12 studies, cystic degeneration was more often associated with hepatocellular hypertrophy or hepatotoxicity. rather than hepatocarcinogenicity. Thus, it is concluded that hepatocarcinogens induce cystic degeneration, not because they are carcinogenic. but because they have other effects on the liver, and that cystic degeneration may be a secondary/reparative change. Cystic degeneration in fish parallels the situation in rats in many respects, yet the existence of the lesion in other species, including man, is not as well supported. Based on the data presented in this review, spontaneous and induced cystic degeneration in rats and fish is not a preneoplastic or neoplastic lesion and risk assessment for man can be based on no-effect levels and safety margins, as for other nonneoplastic adverse effects that have no counterpart in man.

Cutaneous T-cell lymphoma with Sézary syndrome in a dog.

An 8-year-old female spayed Cocker Spaniel mix breed dog was presented with generalized erythroderma, scaling and alopecia. Radiographs of the thorax demonstrated a discrete lung mass which was aspirated using ultrasound guidance and cytological analysis revealed large abnormal lymphocytes. Similar cells were observed in the peripheral blood and in skin biopsies. The cells in the skin biopsies were epidermotropic, indicative of an uncommon cutaneous lymphoma termed cutaneous T cell lymphoma (CTCL), sometimes also called mycosis fungoides. Immunohistochemical staining of a skin biopsy was positive for the CD3 antigen demonstrating that the lymphocyte infiltrate was of a T-cell lineage. The presence of neoplastic lymphocytes in the epidermis and peripheral blood indicate that this is a rare variant of Cutaneous Epidermotropic Lymphoma (CEL) called Sézary syndrome based on nomenclature used in the human literature. An unusual feature of this dog, not seen in previous cases, was the presence of a discrete neoplastic lung mass.