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 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 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 (Reg3γ), a pancreatic acute response protein, was dose-dependently induced in caerulein-treated mice and colocalized with the autophagosomal marker, LC3. This finding supports Reg3γ 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.

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.

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.