Toxicologic Pathology

The 2012 annual National Toxicology Program (NTP) Satellite Symposium, entitled “Pathology Potpourri,” was held in Boston in advance of the Society of Toxicologic Pathology's 31st annual meeting. The goal of the NTP Symposium is to present current diagnostic pathology or nomenclature issues to the toxicologic pathology community. This article presents summaries of the speakers’ presentations, including diagnostic or nomenclature issues that were presented, along with select images that were used for audience voting or discussion. Some lesions and topics covered during the symposium include eosinophilic crystalline pneumonia in a transgenic mouse model; differentiating adrenal cortical cystic degeneration from adenoma; atypical eosinophilic foci of altered hepatocytes; differentiating cardiac schwannoma from cardiomyopathy; diagnosis of cardiac papillary muscle lesions; intrahepatocytic erythrocytes and venous subendothelial hepatocytes; lesions in Rathke’s cleft and pars distalis; pernicious anemia and megaloblastic disorders; embryonic neuroepithelial dysplasia, holoprosencephaly and exencephaly; and INHAND nomenclature for select cardiovascular lesions.

Formaldehyde is a widely used high production chemical that is also released as a byproduct of combustion, off-gassing of various building products, and as a fixative for pathologists and embalmers. What is not often realized is that formaldehyde is also produced as a normal physiologic chemical in all living cells. In 1980, chronic inhalation of high concentrations of formaldehyde was shown to be carcinogenic, inducing a high incidence of nasal squamous cell carcinomas in rats. Some epidemiologic studies have also found increased numbers of nasopharyngeal carcinoma and leukemia in humans exposed to formaldehyde that resulted in formaldehyde being considered a Known Human Carcinogen. This article reviews the data for rodent and human carcinogenicity, early Mode of Action studies, more recent molecular studies of both endogenous and exogenous DNA adducts, and epigenetic studies. It goes on to demonstrate the power of these research studies to provide critical data to improve our ability to develop science-based cancer risk assessments, instead of default approaches. The complexity of constant physiologic exposure to a known carcinogen requires that new ways of thinking be incorporated into determinations of cancer risk assessment for formaldehyde, other endogenous carcinogens, and the role of background endogenous DNA damage and mutagenesis.

Food is not only vital for the health and well-being of any living being, but it is a potential source of harmful chemicals, both natural and man-made. Further complicating this is the fact that most nutrients themselves are potentially toxic when consumed in excess. Deficiencies in some of these same nutrients may cause effects that resemble toxicosis or enhance the toxic potential of other nutrients or exogenous chemicals and drugs. This review discusses some of the nutritional and metabolic mechanisms involved and the implications of excess and deficiency in macronutrients and micronutrients in toxicologic pathology. In addition, we review the adverse effects of ad libitum (AL) overfeeding on metabolic, endocrine, renal, and cardiac diseases, and many cancers and the healthful effects of moderate dietary restriction (DR) in modulating obesity and controlling spontaneous and induced diseases of laboratory animals used in toxicology and carcinogenicity studies for human safety assessment.

Mitochondria, endoplasmic reticulum (ER), cytoplasmic lipid droplets (CLD), and Golgi vesicles use cross talk to control hepatocyte metabolism, growth, and stress. Interpretation of ultrastructural change requires knowledge of how cross talk pathways function, how differential activation of hepatocellular signals influences organelle structure, and how organelles position themselves to become central hubs for stress responses. Mitochondria, by coupling energy production to pathways for protection, form critical platforms for innate signaling. Mitochondrial outer and inner membranes activate channels and signals to translocate peptides that drive oxidative phosphorylation, β-oxidation of fatty acids, and calcium ion (Ca²⁺) flux. In cell stress, mitochondrial signals initiate fusion and fission, reactive oxygen species (ROS) control, autophagy, apoptosis, and senescence. Specialized tethering proteins tie mitochondria to ER to support translocation of metabolites. For Ca²⁺ translocation, ER pores are connected to mitochondrial voltage-dependent anion channels, and for mitochondrial fission, unique membrane proteins pull ER to mitochondria. In toxic injury, cytosolic cytokines translocate to alter metabolism. Toxic effects on ER lipid synthesis lead to Golgi vesicle reduplication and transport of perilipin and other protein cargos into CLDs. How cellular proteostasis, oxidative homeostasis, and ion balance are maintained depend upon the effectiveness of mitochondrial ROS defense responses, unfolded protein responses in mitochondria and ER, and other organelle defenses.

Programmed cell death is physiological when disposing of senescent, dysfunctional, or redundant cells, but pathological if these cells cannot be replaced. Mitochondria help determine cell fate as “gatekeepers” of apoptosis and effectors of cell necrosis. Apoptosis was first described 40 years ago this year. Cell suicide (or the less emotionally charged “programmed cell death”) impacts organism development, normal organ homeostasis, and degenerative (too much cell death) or metaplastic (too little cell death) diseases. The components of apoptosis signaling through mitochondrial targeted Bcl-2 family proteins and activation of the caspase cascade and its downstream proteases and nucleases are well described. More recently, we have realized that there is a parallel cell death pathway, programmed necrosis, in which calcium cross-talk between endoplasmic reticulum and mitochondria causes mitochondrial depolarization, reversal of electron flow through the electron transport chain, and ATP depletion. Since apoptosis and programmed necrosis signaling can occur concurrently in a suicidal cell and are difficult to distinguish using conventional techniques, their relative roles in disease are still being researched and debated. Here, the different molecular mechanisms, effects, and pathophysiological implications of apoptosis and programmed necrosis are reviewed as they relate to heart failure and diabetes mediated by the Bcl-2 family protein, Nix.

The proper folding, assembly, and maintenance of cellular proteins is a highly regulated process and is critical for cellular homeostasis. Multiple cellular compartments have adapted their own systems to ensure proper protein folding, and quality control mechanisms are in place to manage stress due to the accumulation of unfolded proteins. When the accumulation of unfolded proteins exceeds the capacity to restore homeostasis, these systems can result in a cell death response. Unfolded protein accumulation in the endoplasmic reticulum (ER) leads to ER stress with activation of the unfolded protein response (UPR) governed by the activating transcription factor 6 (ATF6), inositol requiring enzyme-1 (IRE1), and PKR-like endoplasmic reticulum kinase (PERK) signaling pathways. Many xenobiotics have been shown to influence ER stress and UPR signaling with either pro-survival or pro-death features. The ultimate outcome is dependent on many factors including the mechanism of action of the xenobiotic, concentration of xenobiotic, duration of exposure (acute vs. chronic), cell type affected, nutrient levels, oxidative stress, state of differentiation, and others. Assessing perturbations in activation or inhibition of ER stress and UPR signaling pathways are likely to be informative parameters to measure when analyzing mechanisms of action of xenobiotic-induced toxicity.

The ICH initiated talks in June 2012 to revise regulatory guidance for carcinogenicity assessment of pharmaceutical products, stimulated in part by a proposal called Negative for Endocrine, Genotoxicity, and Chronic Study Associated Histopathologic Risk Factors for Carcinogenicity in the Rat (NEGCARC) from the Pharmaceutical Research and Manufacturing Association (PhRMA). The 2012 STP Town Hall Meeting focused on the need for change in carcinogenicity assessment strategies for pharmaceuticals. Dr. Todd Bourcier from the Division of Endocrine and Metabolic Products, U.S. FDA and a member of the FDA’s Alternative Carcinogenicity Assessment Committee, was the guest speaker and a panelist. Dr. Bourcier is also one of FDA’s representatives to the ICH S1 Expert Working Group that is discussing changes to regulatory guidelines for carcinogenicity assessment. Drs. Carl Alden and Dan Morton also participated in the panel discussion.

Eplerenone (Inspra®) is an aldosterone receptor antagonist approved for the treatment of hypertension and heart failure after a myocardial infarction. In vitro receptor binding and transactivation studies showed eplerenone had high selectivity for the mineralocorticoid receptor over other steroid receptors (glucocorticoid, androgen, and progesterone). The most sensitive off-target effect of orally administered eplerenone preclinically was prostate atrophy in dogs. Dose-related prostate atrophy was observed at eplerenone dosages ≥15 mg/kg/day for 13 weeks or longer. The no observed adverse effect level (NOAEL) for the prostate effect in dogs was 5 mg/kg/day. The maximal effect was seen by 13 weeks and the atrophy was reversible even after 1 year of daily treatment. An additional study demonstrated dogs with eplerenone-induced prostate atrophy (confirmed by intrarectal ultrasound) had slightly decreased semen volume but no compound-related effects on libido, semen protein content, sperm motility, daily sperm production, or epididymal sperm transit time. Four possible mechanisms for prostate effect were investigated: (1) inhibition of testosterone synthesis and secretion; (2) inhibition of 5α-reductase, the enzyme within the prostate that converts testosterone into the more active growth factor dihydrotestosterone (DHT); (3) competitive antagonism of the androgen receptor; and (4) inhibition of 5α-reductase or competitive antagonism of the androgen receptor by aldosterone, which increased in dogs treated with eplerenone. Data from these studies supported blockade of androgen receptors at suprapharmacological concentrations of eplerenone. Another mineralocorticoid blocker, spironolactone, had greater antiandrogenic activity than eplerenone both in vivo and in vitro, and it has well known clinically significant antiandrogenic effects in humans, whereas eplerenone does not.

Biotherapeutics are expanding the arsenal of therapeutics available for treating and preventing disease. Although initially thought to have limited side effects due to the specificity of their binding, these drugs have now been shown to have potential for adverse drug reactions including effects on peripheral blood cell counts or function. Hematotoxicity caused by a biotherapeutic can be directly related to the activity of the biotherapeutic or can be indirect and due to autoimmunity, biological cascades, antidrug antibodies, or other immune system responses. Biotherapeutics can cause hematotoxicity primarily as a result of cellular activation, cytotoxicity, drug-dependent and independent immune responses, and sequelae from initiating cytokine and complement cascades.  The underlying pathogenesis of biotherapeutic-induced hematotoxicity often is poorly understood. Nonclinical studies have generally predicted clinical hematotoxicity for recombinant cytokines and growth factors.  However, most hematologic liabilities of biotherapeutics are not based on drug class but are species specific, immune-mediated, and of low incidence. Despite the potential for unexpected hematologic toxicity, the risk–benefit profile of most biotherapeutics is favorable; hematologic effects are readily monitorable and managed by dose modification, drug withdrawal, and/or therapeutic intervention.  This article reviews examples of biotherapeutics that have unexpected hematotoxicity in nonclinical or clinical studies. 

Glucagon-like peptide-1 is an incretin hormone from the gastrointestinal tract, which enhances insulin secretion, slows gastric emptying, and reduces food intake. GLP-1 receptor agonists are being developed for Type 2 diabetes mellitus. GLP-1 is rapidly degraded by serum dipeptidyl peptidase IV, so analogues with a prolonged serum half-life are used clinically. Exenatide was the first GLP-1 agonist approved and is a synthetic version of exendin-4 derived from the Gila monster. Liraglutide was approved for clinical use in 2010. GLP-1 receptor agonists have been shown to increase calcitonin secretion and stimulate C-cell hyperplasia and neoplasia in rats and mice of both sexes. Rat C-cells are more sensitive to the effects of GLP-1 agonists than mice. The effects of GLP-1 agonists on C-cell proliferation or neoplasia have not been documented in nonhuman primates or humans. The proliferative C-cell effects may be rodent-specific. GLP-1 receptors have been demonstrated on normal rodent C-cells, but are either not present or occur in low numbers on C-cells of nonhuman primates and humans. Hyperplasia and neoplasia of C-cells in rodents treated with GLP-1 agonists represent a unique example of an on-target species-specific effect that may not have relevance to humans.

Adverse toxicologic effects are categorized as chemical-based, on-target, or off-target effects. Chemical-based toxicity is defined as toxicity that is related to the physicochemical characteristics of a compound and its effects on cellular organelles, membranes, and/or metabolic pathways. On-target refers to exaggerated and adverse pharmacologic effects at the target of interest in the test system. Off-target refers to adverse effects as a result of modulation of other targets; these may be related biologically or totally unrelated to the target of interest. Both the risk assessment and development strategies used for xenobiotics are influenced by the understanding of the mechanism of toxicity. It is imperative that the toxicologic pathologist use the toxicologic and biologic data at hand and literature information on the target to form testable hypotheses related to whether a toxicity is chemical-based, on-target, or off-target. The objective of this session at the 2012 Society of Toxicologic Pathologists Symposium in Boston, Massachusetts, was to discuss chemical-based, on-target, and off-target-based effects and the scientific approaches used to aid in their human risk assessment.

Biologically reactive intermediates formed as endogenous products of various metabolic processes are considered important factors in a variety of human diseases, including Parkinson’s disease and other neurological disorders, diabetes and complications thereof, and other inflammatory-associated diseases. Chemical-induced toxicities are also frequently mediated via the bioactivation of relatively stable organic molecules to reactive electrophilic metabolites. Indeed, chemical-induced toxicities have long been known to be associated with the ability of electrophilic metabolites to react with a variety of targets within the cell, including their covalent adduction to nucleophilic residues in proteins, and nucleotides within DNA. Although we possess considerable knowledge of the various biochemical mechanisms by which chemicals undergo metabolic bioactivation, we understand far less about the processes that couple bioactivation to toxicity. Identifying specific sites within a protein, which are targets for adduction, can provide the initial information necessary to determine whether such adventitious posttranslational modifications significantly alter either protein structure and/or function. To address this problem, we have developed mass spectrometry (MS)-based approaches to identify specific amino acid targets of electrophile adduction (electrophile-binding motifs), coupled with molecular modeling of such adducts, to determine the potential structural and functional consequences. Where appropriate, functional assays are subsequently conducted to assess protein function.

The ability of a chemical to induce mutations has long been a driver in the cancer risk assessment process. The default strategy has been that mutagenic chemicals demonstrate linear cancer dose responses, especially at low exposure levels. In the absence of additional confounding information, this is a reasonable approach, because risk assessment is appropriately considered as being protective of human health. The concept of mode of action has allowed for an opportunity to move off this default position; mutagenicity is now not considered as the driver but rather the mode of action is. In a more precise way, it is the set of key events that define a mode of action that is fundamental in defining the shape of a cancer dose response. A key event is an informative bioindicator of the cancer response and as such should be predictive of the tumor response, at least in a qualitative way. A clear example of the use of key events in cancer risk assessment is for DNA reactive chemicals. A series of such key events is initiated by the production of DNA damage in target cells from direct interaction of the chemical with DNA leading to the production of mutations by misreplication that results in  enhanced cell replication. This enhanced cell replication eventually leads to the development of preneoplastic cells and ultimately overt neoplasms. The response of each of these key events to dose of the chemical can inform the cancer dose–response curve shape. Thus, the dose–response curve for any DNA-reactive chemical can be predicted from knowledge of its mode of action and the behavior of the induced key events.

Hexavalent chromium (Cr(VI)) is a contaminant of water and soil and is a human lung carcinogen. Trivalent chromium (Cr(III)), a proposed essential element, is ingested by humans in the diet and in dietary supplements such as chromium picolinate (CP). The National Toxicology Program (NTP) demonstrated that Cr(VI) is also carcinogenic in rodents when administered in drinking water as sodium dichromate dihydrate (SDD), inducing neoplasms of the oral cavity and small intestine in rats and mice, respectively. In contrast, there was no definitive evidence of toxicity or carcinogenicity following exposure to Cr(III) administered in feed as CP monohydrate (CPM). Cr(VI) readily enters cells via nonspecific anion channels, in contrast to Cr(III), which cannot easily pass through the cell membrane. Extracellular reduction of Cr(VI) to Cr(III), which occurs primarily in the stomach, is considered a mechanism of detoxification, while intracellular reduction is thought to be a mechanism of genotoxicity and carcinogenicity. Tissue distribution studies in additional groups of male rats and female mice demonstrated higher Cr concentrations in tissues following exposure to Cr(VI) compared to controls and Cr(III) exposure at a similar external dose, indicating that some of the Cr(VI) escaped gastric reduction and was distributed systemically. The multiple potential pathways of Cr-induced genotoxicity will be discussed.

Hepatotoxicity is the most common organ injury due to occupational and environmental exposures to industrial chemicals. A wide range of liver pathologies ranging from necrosis to cancer have been observed following chemical exposures both in humans and in animal models. Toxicant-associated fatty liver disease (TAFLD) is a recently named form of liver injury pathologically similar to alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD). Toxicant-associated steatohepatitis (TASH) is a more severe form of TAFLD characterized by hepatic steatosis, inflammatory infiltrate, and in some cases, fibrosis. While subjects with TASH have exposures to industrial chemicals, such as vinyl chloride, they do not have traditional risk factors for fatty liver such as significant alcohol consumption or obesity. Conventional biomarkers of hepatotoxicity including serum alanine aminotransferase activity may be normal in TASH, making screening problematic. This article examines selected chemical exposures associated with TAFLD in human subjects or animal models and concisely reviews the closely related NAFLD and ALD.

There is increasing evidence suggesting links between exposure to environmental toxins and susceptibility to type 2 diabetes mellitus (DM). In this review, we summarize the experimental evidence to support this association that has been noted in many epidemiologic studies. Inflammation in response to particulate matter (PM_2.5) exposure in air pollution represents a common mechanism that may interact with other pro-inflammatory influences in diet and life style to modulate susceptibility to cardiometabolic diseases. The role of innate immune cytokines released from macrophages in the lung is well known. In addition, chemokine triggers in response to air-pollution exposure may mediate a cellular response from the bone marrow/spleen through toll-like receptors (TLRs) and Nucleotide Oligomerization Domain receptors (NLRs) pathways to mediate inflammatory response in organs. Emerging data also seem to support a role for PM_2.5 exposure in endoplasmic reticulum stress-induced apoptosis and in brown adipose tissue dysfunction. Decreased expression of UCP1 in brown adipose tissue may account for reduced thermogenesis providing another link between PM_2.5 and insulin resistance. The implications of an experimental link between air-pollution exposure and type 2 DM are profound as air pollution is a pervasive risk factor throughout the world and even modest alleviation in exposure may provide substantial public health benefits.

Toxicity studies were conducted by the National Toxicology Program (NTP) to provide information on the potential for toxicity from long-term use of commonly used herbal medicines. Here, we review the findings from these NTP toxicology/carcinogenesis 2-year rodent studies of 7 commonly used herbs. In these studies, the individual herb or herbal product was administered to F344/N rats and B6C3F1 mice by oral administration for up to 2 years. The spectrum of carcinogenic responses ranged from no or equivocal evidence for carcinogenic activity (ginseng, milk thistle, and turmeric oleoresin) to a liver tumor response (ginkgo, goldenseal, kava), thyroid tumor response (ginkgo), or an intestinal tumor response (Aloe vera whole leaf nondecolorized extract). Different mechanisms may be involved in the occurrence of liver (ginkgo, goldenseal, and kava kava) and gastrointestinal toxicity (turmeric oleoresin and Aloe vera whole leaf nondecolorized extract), while the toxic lesion is the same. The results from these hazard identification toxicity/carcinogenesis studies along with those from ongoing National Institute of Health clinical trials of herbal medicines provide more complete information on the risks and benefits from herbal medicine use in the general population.

The goal of this article is to evaluate a recently published subchronic inhalation study with carbon nanofibers in rats and discuss the importance of a weight-of-evidence (WOE) framework for determining no adverse effect levels (NOAELs). In this Organization for Economic Cooperation and Development (OECD) 413 guideline inhalation study with VGCF™-H carbon nanofibers (CNFs), rats were exposed to 0, 0.54, 2.5 or 25 mg/m³ CNF for 13 weeks. The standard toxicology experimental design was supplemented with bronchoalveolar lavage (BAL) and respiratory cell proliferation (CP) endpoints. BAL fluid (BALF) recovery of inflammatory cells and mediators (i.e., BALF– lactate dehydrogenase [LDH], microprotein [MTP], and alkaline phosphatase [ALKP] levels) were increased only at 25 mg/m³, 1 day after exposure. No differences versus control values in were measured at 0.54 or 2.5 mg/m³ exposure concentrations for any BAL fluid endpoints. Approximately 90% (2.5 and 25 mg/m³) of the BAL-recovered macrophages contained CNF. CP indices at 25 mg/m³ were increased in the airways, lung parenchyma, and subpleural regions, but no increases in CP versus controls were measured at 0.54 or 2.5 mg/m³. Based upon histopathology criteria, the NOAEL was set at  0.54 mg/m³, because at 2.5 mg/m³, “minimal cellular inflammation” of the airways/lung parenchyma was noted by the study pathologist; while the 25 mg/m³ exposure concentration produced slight inflammation and occasional interstitial thickening. In contrast, none of the more sensitive pulmonary biomarkers such as BAL fluid inflammation/cytotoxicity biomarkers or CP turnover results at 2.5 mg/m³ were different from air-exposed controls. Given the absence of convergence of the histopathological observations versus more quantitative measures at 2.5 mg/m³, it is recommended that more comprehensive guidance measures be implemented for setting adverse effect levels in (nano)particulate, subchronic inhalation studies including a WOE approach for establishing no adverse effect levels; and a suggestion that some findings should be viewed as normal physiological adaptations (e.g., normal macrophage phagocytic responses—minimal inflammation) to long-term particulate inhalation exposures.

Nanotechnology involves technology, science, and engineering in dimensions less than 100 nm. A virtually infinite number of potential nanoscale products can be produced from many different molecules and their combinations. The exponentially increasing number of nanoscale products will solve critical needs in engineering, science, and medicine. However, the virtually infinite number of potential nanotechnology products is a challenge for toxicologic pathologists. Because of their size, nanoparticulates can have therapeutic and toxic effects distinct from micron-sized particulates of the same composition. In the nanoscale, distinct intercellular and intracellular translocation pathways may provide a different distribution than that obtained by micron-sized particulates. Nanoparticulates interact with subcellular structures including microtubules, actin filaments, centrosomes, and chromatin; interactions that may be facilitated in the nanoscale. Features that distinguish nanoparticulates from fine particulates include increased surface area per unit mass and quantum effects. In addition, some nanotechnology products, including the fullerenes, have a novel and reactive surface. Augmented microscopic procedures including enhanced dark-field imaging, immunofluorescence, field-emission scanning electron microscopy, transmission electron microscopy, and confocal microscopy are useful when evaluating nanoparticulate toxicologic pathology. Thus, the pathology assessment is facilitated by understanding the unique features at the nanoscale and the tools that can assist in evaluating nanotoxicology studies.

The peroxisome proliferator–activated receptor (PPAR) family of nuclear hormone transcription factors (PPARα, PPARβ/δ, and PPARγ) is regulated by a wide array of ligands including natural and synthetic chemicals. PPARs have important roles in control of energy metabolism and are known to influence inflammation, differentiation, carcinogenesis, and chemical toxicity. As such, PPARs have been targeted as therapy for common disorders such as cancer, metabolic syndrome, obesity, and diabetes. The recent application of metabolomics, or the global, unbiased measurement of small molecules found in biofluids, or extracts from cells, tissues, or organisms, has advanced our understanding of the varied and important roles that the PPARs have in normal physiology as well as in pathophysiological processes. Continued development and refinement of analytical platforms, and the application of new bioinformatics strategies, have accelerated the widespread use of metabolomics and have allowed further integration of small molecules into systems biology. Recent studies using metabolomics to understand PPARα function, as well as to identify PPARα biomarkers associated with drug efficacy/toxicity and drug-induced liver injury, will be discussed.