Jon C. Cook

PPARα Agonist-Induced Rodent Tumors: Modes of Action and Human Relevance

Widely varied chemicals—including certain herbicides, plasticizers, drugs, and natural products—induce peroxisome proliferation in rodent liver and other tissues. This phenomenon is characterized by increases in the volume density and fatty acid oxidation of these organelles, which contain hydrogen peroxide and fatty acid oxidation systems important in lipid metabolism. Research showing that some peroxisome proliferating chemicals are nongenotoxic animal carcinogens stimulated interest in developing mode of action (MOA) information to understand and explain the human relevance of animal tumors associated with these chemicals. Studies have demonstrated that a nuclear hormone receptor implicated in energy homeostasis, designated peroxisome proliferator-activated receptor alpha (PPARα), is an obligatory factor in peroxisome proliferation in rodent hepatocytes. This report provides an in-depth analysis of the state of the science on several topics critical to evaluating the relationship between the MOA for PPARα agonists and the human relevance of related animal tumors. Topics include a review of existing tumor bioassay data, data from animal and human sources relating to the MOA for PPARα agonists in several different tissues, and case studies on the potential human relevance of the animal MOA data. The summary of existing bioassay data discloses substantial species differences in response to peroxisome proliferators in vivo, with rodents more responsive than primates. Among the rat and mouse strains tested, both males and females develop tumors in response to exposure to a wide range of chemicals including DEHP and other phthalates, chlorinated paraffins, chlorinated solvents such as trichloroethylene and perchloroethylene, and certain pesticides and hypolipidemic pharmaceuticals. MOA data from three different rodent tissues—rat and mouse liver, rat pancreas, and rat testis—lead to several different postulated MOAs, some beginning with PPARα activation as a causal first step. For example, studies in rodent liver identified seven “key events,” including three “causal events”—activation of PPARα, perturbation of cell proliferation and apoptosis, and selective clonal expansion—and a series of associative events involving peroxisome proliferation, hepatocyte oxidative stress, and Kupffer-cell-mediated events. Similar in-depth analysis for rat Leydig-cell tumors (LCTs) posits one MOA that begins with PPARα activation in the liver, but two possible pathways, one secondary to liver induction and the other direct inhibition of testicular testosterone biosynthesis. For this tumor, both proposed pathways involve changes in the metabolism and quantity of related hormones and hormone precursors. Key events in the postulated MOA for the third tumor type, pancreatic acinar-cell tumors (PACTs) in rats, also begin with PPARα activation in the liver, followed by changes in bile synthesis and composition. Using the new human relevance framework (HRF) (see companion article), case studies involving PPARα-related tumors in each of these three tissues produced a range of outcomes, depending partly on the quality and quantity of MOA data available from laboratory animals and related information from human data sources.

Hemangiosarcoma in Rodents: Mode-of-Action Evaluation and Human Relevance

Although rarely occurring in humans, hemangiosarcomas (HS) have become important in evaluating the potential human risk of several chemicals, including industrial, agricultural, and pharmaceutical agents. Spontaneous HS arise frequently in mice, less commonly in rats, and frequently in numerous breeds of dogs. This review explores knowledge gaps and uncertainties related to the mode of action (MOA) for the induction of HS in rodents, and evaluates the potential relevance for human risk. For genotoxic chemicals (vinyl chloride and thorotrast), significant information is available concerning the MOA. In contrast, numerous chemicals produce HS in rodents by nongenotoxic, proliferative mechanisms. An overall framework is presented, including direct and indirect actions on endothelial cells, paracrine effects in local tissues, activation of bone marrow endothelial precursor cells, and tissue hypoxia. Numerous obstacles are identified in investigations into the MOA for mouse HS and the relevance of the mouse tumors to humans, including lack of identifiable precursor lesions, usually late occurrence of the tumors, and complexities of endothelial biology. This review proposes a working MOA for HS induced by nongenotoxic compounds that can guide future research in this area. Importantly, a common MOA appears to exist for the nongenotoxic induction of HS, where there appears to be a convergence of multiple initiating events (e.g., hemolysis, decreased respiration, adipocyte growth) leading to either dysregulated angiogenesis and/or erythropoiesis that results from hypoxia and macrophage activation. These later events lead to the release of angiogenic growth factors and cytokines that stimulate endothelial cell proliferation, which, if sustained, provide the milieu that can lead to HS formation.

Evaluation of Tier I screening approaches for detecting endocrine-active compounds (EACs).

In 1996, Congress passed legislation requiring the U.S. Environmental Protection Agency (EPA) to implement screening/testing strategies for endocrine-active compounds (EACs). In response, EPA convened the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) to advise the agency on a strategy to screen and test xenobiotics for endocrine disruption. EDSTAC completed their charter in 1998 by recommending a tiered screening and testing scheme to evaluate compounds for their potential to act as agonists or antagonists to the estrogen or androgen receptors, steroid biosynthesis inhibitors, or their ability to alter thyroid function. For Tier I, the EDSTAC-recommended screening battery comprised eight different assays, but EDSTAC also proposed two alternative batteries that were deemed worthy of further evaluation. The challenge currently confronting EPA is to choose among the Tier I screening options and then to standardize protocols, validate the assays, and determine the criteria for judging a compound as positive or negative in the battery. The purpose of the current review is to: (1) provide an overview of the three EDSTAC options, (2) evaluate the data currently available for the individual assays of the three EDSTAC options and discuss the strengths and limitations of each, and (3) provide a final recommendation for a Tier I screen based on the experiences of the authors who have used all of the individual assays under consideration by EDSTAC. The goal of this report is not to provide an exhaustive historical review of each assay, but rather to summarize some of the more relevant data from available published reports as it relates to current proposed study designs for those particular assays. Based on the current data, a Tier I screening battery consisting of in vitro receptor binding assays, a 3-day uterotrophic assay, and a 15-day intact male assay are recommended as the preferred approach on which future validation efforts should be focused. This screening approach is a mode-of-action screen that will identify specific types of endocrine activity. Because it utilizes many endpoints from the same test animals (i.e., it integrates), it is the most cost-effective and efficient option in terms of animal usage. The mode-of-action screening approach advances scientific understanding and is preferred over other options based on apical tests, as these essentially are reproductive effects screens that are not necessarily specific for endocrine activity. Because Tier II tests include the critical apical endpoints used in the pubertal models, a mode-of-action approach provides complementary rather than redundant data. By identifying the potential mode of action, critical endpoints can be included in Tier II studies that will be used to define dose-response curves and no observed adverse effect levels (NOAELs)/no observed effect levels (NOELs) for the compound.

The Role of Hypoxia in 2-Butoxyethanol–Induced Hemangiosarcoma

To understand the molecular mechanisms underlying compound-induced hemangiosarcomas in mice, and therefore, their human relevance, a systems biology approach was undertaken using transcriptomics and Causal Network Modeling from mice treated with 2-butoxyethanol (2-BE). 2-BE is a hemolytic agent that induces hemangiosarcomas in mice. We hypothesized that the hemolysis induced by 2-BE would result in local tissue hypoxia, a well-documented trigger for endothelial cell proliferation leading to hemangiosarcoma. Gene expression data from bone marrow (BM), liver, and spleen of mice exposed to a single dose (4 h) or seven daily doses of 2-BE were used to develop a mechanistic model of hemangiosarcoma. The resulting mechanistic model confirms previous work proposing that 2-BE induces macrophage activation and inflammation in the liver. In addition, the model supports local tissue hypoxia in the liver and spleen, coupled with increased erythropoeitin signaling and erythropoiesis in the spleen and BM, and suppression of mechanisms that contribute to genomic stability, events that could be contributing factors to hemangiosarcoma formation. Finally, an immunohistochemistry method (Hypoxyprobe) demonstrated that tissue hypoxia was present in the spleen and BM. Together, the results of this study identify molecular mechanisms that initiate hemangiosarcoma, a key step in understanding safety concerns that can impact drug decision processes, and identified hypoxia as a possible contributing factor for 2-BE–induced hemangiosarcoma in mice.

ROLE OF PROLACTIN IN CHLORO-S-TRIAZINE RAT MAMMARY TUMORIGENESIS

Chloro-S-triazine herbicides [cyanazine (CZ), atrazine (AZ), simazine (SZ)] increase mammary tumors in Crl:CD® BR rats but not in F-344 rats or in mice. A nongenotoxic mechanism was investigated since the chloro-S-triazines are negative in short-term tests for genotoxicity. An in vivo battery was used to assess the chloro-S-triazines for estrogenic activity or for their ability to increase prolactin (PRL) levels, both of which play important roles in enhancing mammary gland tumorigenesis in rodents. Ovariectomized (OVX) female rats were treated with AZ, CZ, SZ, or three CZ metabolites for 4 days via intraperitoneal injection. The pattern of responses between the chloro-S-triazines and four controls (estradiol, estriol, haloperidol, reserpine) was compared. For the 6 endpoints examined, the responses from rats treated with AZ, CZ, SZ, and the metabolites of CZ most closely matched the responses from the reserpine-treated rats (a PRL rather than estrogenic mechanism). In addition, AZ, CZ, and SZ were tested in several other in vitro models (estrogen/biogenic amine receptor competition assays and a yeast-expressed human estrogen receptor transcription assay) as well as an in vivo 24h time-course experiment to characterize the CZ-induced increases in PRL levels. AZ, CZ, and SZ are not estrogen receptor (ER) activating compounds based on yeast transactivation and receptor competition data. CZ and AZ demonstrated marginal competition (at mM levels) to the D and α2 adrenergic receptors. Ligands to the D2 receptor, but not the α2 adrenergic receptor, are known to induce mammary tumors. CZ was also found to produce elevated PRL levels in a time-course similar to that seen with reserpine and haloperidol. Overall, the pattern of responses obtained with the chloro-S-triazines most closely matched the responses observed for reserpine. Taken together, these data suggest chloro-S-triazine-induced mammary tumors in rats are mediated through a PRL mechanism, which is thought to be of low relevance to humans.

Exposure to Pharmaceuticals Present in the Environment

Pharmaceuticals have been detected in the environment with increasing frequency since the late 1990s. The concentrations detected are low. For example, the average concentration of a pharmaceutical detected in surface waters is 0.042 parts per billion (μg/L) when the value for samples reported to contain nondetectable levels of an analyte is assumed to be 0 and 0.151 when these samples without detection are not included in the averaging. The bioactivity of pharmaceuticals has led to concerns that organisms in the environment may be impacted by ongoing exposure as a result of continuing patient use. The appropriateness of the existing environmental science and data for answering key questions pertaining to the fate and effect of bioactive pharmaceuticals has been questioned. The objectives of this article are to describe the sources of human pharmaceuticals detected in the environment, provide a detailed summary of the worldwide detection data, evaluate the potential significance of the environmental concentrations reported, and propose a mechanistic approach to the exposure analysis for environmental risk assessment. Open scientific questions are presented.

Evaluation of Expression Profiles of Hematopoietic Stem Cell, Endothelial Cell, and Myeloid Cell Antigens in Spontaneous and Chemically Induced Hemangiosarcomas and Hemangiomas in Mice

It is unclear whether the process of spontaneous and chemically induced hemangiosarcoma and hemangioma formation in mice involves the transformation of differentiated endothelial cells (ECs) or recruitment of multipotential bone marrow–derived hematopoietic stem cells or endothelial progenitor cells (EPCs), which show some degree of endothelial differentiation. In the present study, immunohistochemical staining for hematopoietic stem cell markers (CD45 and CD34), EC markers (vascular endothelial growth factor receptor 2 [VEGFR2], CD31, and factor VIII–related antigen), and a myeloid lineage marker (CD14) was employed to better define the origin of hemangiosarcomas and hemangiomas in mice. Staining was negative for CD45, factor VIII–related antigen, and CD14 and positive for CD34, VEGFR2, and CD31, indicating that mouse hemangiosarcomas and hemangiomas are composed of cells derived from EPCs expressing CD34, VEGFR2, and CD31 but not factor VIII–related antigen. The lack of CD45 expression suggests that mouse vascular tumors may arise from EPCs that are at a stage later than hematopoietic stem cells. Since factor VIII–related antigen expression is known to occur later than CD31 expression in EPCs, our observations may indicate that these tumor cells are arrested at a stage prior to complete differentiation. In addition, myeloid lineage cells do not appear to contribute to hemangiosarcoma and hemangioma formation in mice.

Combined Treatment Potentiates the Developmental Toxicity of Ibuprofen and Acetazolamide in Rats

Aspirin (ASA), an irreversible cyclooxygenase (COX) inhibitor, induces ventricular septal defect (VSD) and diaphragmatic hernia (DH) in rat fetuses when administered on gestation days (GDs) 9–10, a critical period for cardiovascular (CV) and midline development. Evaluation of a spectrum of nonsteroidal antiinflammatory drugs (NSAIDs; reversible COX inhibitors) showed that while some NSAIDs induced VSD in rats, none of the NSAIDs evaluated produced DH. In addition to inhibiting COX, ASA also inhibits carbonic anhydrase. The purpose of this study was to determine whether concurrent inhibition of COX and carbonic anhydrase would produce a teratogenic profile that includes both VSD and DH. To inhibit both COX and carbonic anhydrase, ibuprofen (COX inhibitor) and acetazolamide (carbonic anhydrase inhibitor) were coadministered on GDs 9–10. Groups of 20 female Crl:CD(SD)IGS BR rats were given either 300 mg kg− 1 day− 1 ibuprofen, 1000 mg kg− 1 day− 1 acetazolamide, or both (combination of ibuprofen and acetazolamide). Fetuses were evaluated on GD 21 for external and visceral development. Ibuprofen induced VSD in 3.7% of fetuses per litter; no defects in appendicular skeletal development were noted. Acetazolamide induced VSD in 5.9% of the fetuses per litter and appendicular defects in 41% of the fetuses per litter. Coadministration of ibuprofen and acetazolamide produced VSD in 18.7% of the fetuses per litter and appendicular defects in 77% of the fetuses per litter; however, there were no DH. Therefore, while concurrent inhibition of COX and carbonic anhydrase did not produce DH, potentiation was noted for the induction of VSD and appendicular anomalies.

Pregabalin Induces Hepatic Hypoxia and Increases EndothelialCell Proliferation in Mice, a Process Inhibited by DietaryVitamin E Supplementation

The preceding article identified key components of pregabalins mode of action on nongenotoxic hemangiosarcoma formation in mice, including increased serum bicarbonate leading to decreased respiratory rate, increased blood pH, increased venous oxygen saturation, increased vascular endothelial growth factor and basic fibroblast growth factor expression, increased hepatic vascular endothelial growth factor receptor 2 expression, and increased iron-laden macrophages. Increased platelet count and platelet activation were early, species-specific biomarkers in mice. Dysregulated erythropoiesis, macrophage activation, and elevations of tissue growth factors were consistent with the unified mode of action for nongenotoxic hemangiosarcoma recently proposed at an international hemangiosarcoma workshop (Cohen, S. M., Storer, R. D., Criswell, K. A., Doerrer, N. G., Dellarco, V. L., Pegg, D. G., Wojcinski, Z. W., Malarkey, D. E., Jacobs, A. C., Klaunig, J. E., et al. (2009). Hemangiosarcoma in rodents: Mode-of-action evaluation and human relevance. Toxicol. Sci. 111, 4-18). In this article, we present evidence that pregabalin induces hypoxia and increases endothelial cell (EC) proliferation in a species-specific manner. Dietary administration of pregabalin produced a significant 35% increase in an immunohistochemical stain for hypoxia (Hypoxyprobe) in livers from pregabalin-treated mice. Increased Hypoxyprobe staining was not observed in the liver, bone marrow, or spleen of rats, supporting the hypothesis that pregabalin produces local tissue hypoxia in a species-specific manner. Transcriptional analysis supports that rats, unlike mice, adapt to pregabalin-induced hypoxia. Using a dual-label method, increased EC proliferation was observed as early as 2 weeks in mouse liver and 12 weeks in bone marrow following pregabalin administration. These same assays showed decreased EC proliferation in hepatic ECs of rats, further supporting species specificity. Dietary supplementation with vitamin E, which is known to have antioxidant and antiangiogenic activity, inhibited pregabalin-induced increases in mouse hepatic EC proliferation, providing confirmatory evidence for the proposed mode of action and its species-specific response.

Mode-of-Action Associated With Development of Hemangiosarcoma in Mice Given Pregabalin, and Assessment of Human Relevance

Pregabalin increased the incidence of hemangiosarcomas in carcinogenicity studies of 2-year mice but was not tumorigenic in rats. Serum bicarbonate increased within 24 h of pregabalin administration in mice and rats. Rats compensated appropriately, but mice developed metabolic alkalosis and increased blood pH. Local tissue hypoxia and increased endothelial cell proliferation were also confirmed in mice alone. The combination of hypoxia and sustained increases in endothelial cell proliferation, angiogenic growth factors, dysregulated erythropoiesis, and macrophage activation is proposed as the key event in the mode of action (MOA) for hemangiosarcoma formation. Hemangiosarcomas occur spontaneously in untreated control mice but occur only rarely in humans. The International Programme on Chemical Safety and International Life Sciences Institute developed a Human Relevance Framework (HRF) analysis whereby presence or absence of key events can be used to assess human relevance. The HRF combines the MOA with an assessment of biologic plausibility in humans to assess human relevance. This manuscript compares the proposed MOA with Hill criteria, a component of the HRF, for strength, consistency, specificity, temporality, and dose response, with an assessment of key biomarkers in humans, species differences in response to disease conditions, and spontaneous incidence of hemangiosarcoma to evaluate human relevance. Lack of key biomarker events in the MOA in rats, monkeys, and humans supports a species-specific process and demonstrates that the tumor findings in mice are not relevant to humans at the clinical dose of pregabalin. Based on this collective dataset, clinical use of pregabalin would not pose an increased risk for hemangiosarcoma to humans.